Cell Constriction: Contractile Role of Microfilaments in Division and Development

The role of microfilaments in causing cell constrictions isdiscussed from a comparative point of view. Morphologicallysimilar microfilaments occur in the contractile ring of dividingcells and in the apices of neural plate cells during neurulation.New evidence is presented regarding the distribution and orientationof apical microfilaments in neural plates of chicks and salamanderembryos. These findings complement what is known about neurulationin frogs and cell cleavage in a variety of cells. In all cases,cell constriction occurs precisely and exclusively at thoseplanes in which circular arrays of microfilaments are found.A sliding mechanism of microfilament contractility is discussed,as are possible mechanisms involved in filament alignment. Attentionis given to the cell surface as a substratum for microfilamentassembly. New evidence is presented regarding the early morphologicaldetermination of the neural plate in Xenopus, even before microfilamentsare clearly evident or invagination begins.     

微丝骨架在植物细胞信号转导中的作用

文章从小G蛋白、离子浓度和肌醇磷脂信号系统等方面阐述植物细胞微丝骨架与细胞信号转导的关系.     

Role of Actin Microfilaments in Black Creek Canal Virus Morphogenesis

We have investigated the involvement of cytoskeletal proteins in the morphogenesis of Black Creek Canal virus (BCCV), a New World hantavirus. Immunofluorescent staining of BCCV-infected cells revealed a filamentous pattern of virus antigen, the appearance of which was sensitive to treatment with cytochalasin D, an actin microfilament-depolymerizing drug. Double immunofluorescence staining of BCCV-infected Vero cells with anti-BCCV nucleocapsid (N) monoclonal antibody and phalloidin revealed a colocalization of the BCCV N protein with actin microfilaments. A similar, though less prominent, filamentous pattern was observed in BHK21 cells transiently expressing the BCCV N protein alone but not in cells expressing the BCCV G1 and G2 glycoproteins. Moreover, the association of the N protein with actin microfilaments was confirmed by coimmunoprecipitation with β-actin-specific antibody. Treatment of the BCCV-infected Vero cells at 3 days postinfection with cytochalasin D decreased the yield of released BCCV by 94% relative to the yield from untreated cells. Pretreatment of Vero cells with cytochalasin D prior to and during BCCV adsorption and entry had no effect on the outcome of virus production. These results indicate that actin filaments may play an important role in hantavirus assembly and/or release.     

A Role for Myocilin in Receptor-Mediated Endocytosis

Myocilin is a broadly expressed protein that when mutated uniquely causes glaucoma. While no function has been ascribed to explain focal disease, some properties of myocilin are known. Myocilin is a cytoplasmic protein that also localizes to vesicles specifically as part of a large membrane-associated complex with properties similar to the SNARE machinery that function in vesicle fusion. Its role in vesicle dynamics has not been detailed, however myocilin intersects with the endocytic compartment at the level of the multivesicular body. Since internalized GPCRs are sorted in the multivesicular body, we investigated whether myocilin functions in ligand-dependent GPR143 endocytosis. Using recombinant systems we found that the kinetics of myocilin recruitment to biotinylated membrane proteins was similar to that of arrestin-3. We also co-localized myocilin with GPR143 and Arrestin-2 by confocal microscopy. However, wild-type myocilin differed significantly in its association kinetics and co-localization with internalized proteins from mutant myocilin (P370L or T377M). Moreover, we found that myocilin bound to the cytoplasmic tail of GPR143, an interaction mediated by its amino terminal helix-turn-helix domain. Hydrodynamic analyses show that the myocilin-GPR143 protein complex is >158 kD and stable in 500 mM KCl, but not 0.1% SDS. Collectively, data indicate that myocilin is recruited to the membrane compartment, interacting with GPCR proteins during ligand-mediated endocytosis and that GPCR signaling underlies pathology in myocilin glaucoma.     

Role of Small Heat Shock Proteins in the Remodeling of Actin Microfilaments

Biochemistry (Moscow) - Small heat shock proteins (sHsps) play an important role in the maintenance of proteome stability and, particularly, in stabilization of the cytoskeleton and cell...     

The Role of Endocytosis during Morphogenetic Signaling

Morphogens are signaling molecules that are secreted by a localized source and spread in a target tissue where they are involved in the regulation of growth and patterning. Both the activity of morphogenetic signaling and the kinetics of ligand spreading in a tissue depend on endocytosis and intracellular trafficking. Here, we review quantitative approaches to study how large-scale morphogen profiles and signals emerge in a tissue from cellular trafficking processes and endocytic pathways. Starting from the kinetics of endosomal networks, we discuss the role of cellular trafficking and receptor dynamics in the formation of morphogen gradients. These morphogen gradients scale during growth, which implies that overall tissue size influences cellular trafficking kinetics. Finally, we discuss how such morphogen profiles can be used to control tissue growth. We emphasize the role of theory in efforts to bridge between scales.A fundamental challenge in biology is to understand how morphologies and complex patterns form in multicellular systems by the collective organization of many cells. Cells divide and undergo apoptosis, and they communicate via signaling pathways that use molecules as information carriers. In tissues, large-scale patterns of gene expression emerge from the coordinated signaling activity and response of many cells. The establishment of such patterns is often guided by long-range concentration profiles of morphogens. Cell divisions and cell rearrangements must be coordinated over large distances to achieve specific tissue sizes and shapes. To unravel how molecular processes and interactions can eventually be responsible for the formation of structures and patterns in tissues during development, it is important to study processes at different scales and understand how different levels of organization are connected. Such an approach becomes strongest if it involves a combination of quantitative experimental studies with theory.In the present article, we discuss several such approaches on different scales with a particular emphasis on theory. Starting from the kinetic and dynamic properties of endosomal networks inside a cell, we discuss transport processes in a tissue that can be related to kinetic trafficking parameters. Such transport processes are then responsible for the formation of graded morphogen concentration profiles. To permit scalable patterns in tissues of different sizes, it has been suggested that morphogen gradients scale during growth. This can be achieved on the tissue level by feedback systems that are sensitive to tissue size and regulate, for example, morphogen degradation. Finally, morphogen gradients that scale with tissue size can provide a system to robustly organize cell division in a large tissue and generate homogeneous growth. Theory can play an important role to bridge scales and understand how molecular and cellular processes can control pattern formation and tissue growth on larger scales.Morphogens are signaling molecules that are secreted in specific regions of developing tissues and can induce signaling activity far from their source. They typically form graded concentration profiles and therefore endow cells with positional information (cells can obtain information about their position in a tissue). Thus, they can guide cells to differentiate into complex morphological patterns. Morphogens also control cell growth and cell division. Because they control both patterning and growth, they may play a key role to coordinate these two processes. Such coordination is important because the size of morphological patterns must adjust during growth, whereas growth influences such patterns. A well-studied morphogen is Decapentaplegic (Dpp), which controls morphogenesis in the imaginal wing disc of developing Drosophila. Consequently, mutations in Dpp or defects in the trafficking pathways that control its graded concentration profiles and signaling affect the formation and structure of the adult wing.The study of morphogens was traditionally approached from a genetic perspective: Which gene products behave like morphogens? Which mutants affect patterning and growth? The realization that morphogens typically operate by a gradient of concentration raised the question of how morphogen gradients are generated. It became clear that the cellular trafficking of morphogens is a key issue for the generation of morphogen profiles. Morphogens are secreted ligands that bind receptors in the plasma membrane. The secretion of the ligands and the concentrations of receptor, ligand, and receptor/ligand complex at the plasma membrane are governed by their trafficking in the cell by vesicular transport. In particular, it was shown that trafficking through the endocytic pathway has an important impact on the formation of morphogen gradients (reviewed in Gonzalez-Gaitan 2003; see Bökel and Brand 2014). This is, to a large extent, how the cells respond to morphogens and contribute to set their local concentrations. To understand functions of morphogens in a tissue, we need to study how the gradient is formed. This, in turn, requires insights into morphogen trafficking through the endocytic pathway. The problem of morphogen behavior, therefore, becomes a problem spanning several levels of complexity: the organ level, the tissue level, the cell level, the organelle level, and the molecular level. Theoretical approaches motivated by physics combined with quantitative experimental approaches provide an ideal framework to understand how these different levels of complexity are intertwined.Two recent discoveries highlighted such integration. (1) The observation that profiles of the morphogen Dpp scale during growth, which implies that the rate of Dpp degradation mediated by the endocytic pathway of each of the cells in the tissue depends on the size of the overall tissue. This suggests that two levels of complexity are linked because cellular trafficking receives cues about the global tissue size. (2) As a result of the changes of the degradation rate that leads to gradient scaling, cells receive an increasing level of signaling. This, in turn, can be used by the cells to decide when to divide. This regulation again involves two levels of complexity because regulation at the endocytic pathway determines the growth properties of the tissue and, ultimately, its final size.In the following, we discuss quantitative approaches to study cellular signaling processes on different scales. Here, the aim is to understand how patterns on large scales can emerge during development from molecular processes and signaling pathways that involve endocytosis and cellular trafficking. We begin by describing trafficking of ligands in the endocytic pathway. We then consider the situation of a morphogen ligand and its impact in gradient formation. Subsequently, we discuss how gradient scaling might be realized. Finally, we discuss how such scaling processes play an important role in the regulation of morphogenetic growth.     

Amphiphysin Heterodimers: Potential Role in Clathrin-mediated Endocytosis

Amphiphysin (Amph) is a src homology 3 domain-containing protein that has been implicated in synaptic vesicle endocytosis as a result of its interaction with dynamin. In a screen for novel members of the amphiphysin family, we identified Amph2, an isoform 49% identical to the previously characterized Amph1 protein. The subcellular distribution of this isoform parallels Amph1, both being enriched in nerve terminals. Like Amph1, a role in endocytosis at the nerve terminal is supported by the rapid dephosphorylation of Amph2 on depolarization. Importantly, the two isoforms can be coimmunoprecipitated from the brain as an equimolar complex, suggesting that the two isoforms act in concert. As determined by cross-linking of brain extracts, the Amph1–Amph2 complex is a 220- to 250-kDa heterodimer. COS cells transfected with either Amph1 or Amph2 show greatly reduced transferrin uptake, but coexpression of the two proteins rescues this defect, supporting a role for the heterodimer in clathrin-mediated endocytosis. Although the src homology 3 domains of both isoforms interact with dynamin, the heterodimer can associate with multiple dynamin molecules in vitro and activates dynamin’s GTPase activity. We propose that it is an amphiphysin heterodimer that drives the recruitment of dynamin to clathrin-coated pits in endocytosing nerve terminals.     

Phosphatidic Acid Plays a Regulatory Role in Clathrin-mediated Endocytosis

Clathrin-mediated endocytosis (CME) is the main route of internalization of receptor-ligand complexes. Relatively little is known about the role of specific lipids in CME, in particular that of phosphatidic acid (PA). We examined the effect of altering cellular PA levels on CME by manipulating the activities and/or levels of either phospholipase D (PLD1 and PLD2) or diacylglycerol kinase (DGK), two enzyme classes involved in PA production. DGK inhibition resulted in a dramatic reduction of cellular PA, measured directly using an enzyme-coupled reaction, which resulted in a decreased rate of EGFR internalization measured biochemically. This corresponded to a decreased rate of clathrin-coated pit (CCP) initiation and increased lifetimes of productive CCPs, as determined by quantitative live-cell total internal reflection fluorescence microscopy. Unexpectedly, PLD inhibition caused an increase in cellular PA, suggesting that PLD activity negatively regulates PA synthesis by other more productive pathways. Consistent with opposite effects on cellular PA levels, PLD inhibition had opposite effects on EGFR internalization and CCP dynamics, compared with DGK inhibition. Importantly, the constitutive internalization of transferrin receptors was unaffected by either treatment. These findings demonstrate that PA plays a regulatory rather than obligatory role in CME and differentially regulates ligand-stimulated CME of EGFR.     

Cost and Advantage of Soil Channel Formation by Contractile Roots in Successful Plant Movement

In most cases, contractile roots not only produce a pullingforce on an underground plant (bulb, corm), but also push awaythe substratum and create a soil channel in which plant movementis made easier. Two specially-constructed experimental set-upsallowed the pulling and pushing activity of contractile rootsin underground plant movement to be measured under identicalstandard conditions. Our results show that root channel formationis always advantageous for the accomplishment of movement. Theextent of channel formation in relation to the size of the bulbor corm being moved was calculated as a percentage channel effect.A value of 100% indicates a channel effect sufficient to allowplant movement with no resistance. For smaller distances moved(20-30 mm) a small channel effect (10-40%) was found to be veryefficient. For more extensive movement (> 50 mm) a 100% channeleffect seems to be the optimum energetically. These systemsof movement commonly exist in nature.Copyright 1995, 1999 AcademicPress Contractile root, root contraction, soil channel, channel effect, plant movement     

On the Role of Microfilaments In Cell-Shape-Mediated Growth Control of Lens Epithelial Cells

With regard to the fact that, in anchorage-dependent lens epithelial cells, DNA synthesis can be switched on and off by cell flattening and cell rounding, respectively, the state of the microfilaments has been followed by labelling actin with FL-phalloidin during cell-shape alterations. Cell flattening proved to be accompanied by both a structural organization of actin filaments into stress fibres and an enlargement of the area of the cell nucleus. Cell rounding, on the other hand, caused the microfilament bundles to disappear and the area of the nucleus to become smaller. From the time course of the inhibition of DNA synthesis by cytochalasin B, it was inferred that functionally intact microfilaments are required for the entrance of the cells into DNA synthesis but not for the maintenance of ongoing DNA synthesis. the assumption has been made that the tension, generated by microfilaments during cell spreading, will affect the state of the plasma membrane as well as the shape and the structure of the nucleus, which in turn seems to be preparatory for cells to enter the cycle.     

The Role of Macrophage Polarization in Infectious and Inflammatory Diseases

Macrophages, found in circulating blood as well as integrated into several tissues and organs throughout the body, represent an important first line of defense against disease and a necessary component of healthy tissue homeostasis. Additionally, macrophages that arise from the differentiation of monocytes recruited from the blood to inflamed tissues play a central role in regulating local inflammation. Studies of macrophage activation in the last decade or so have revealed that these cells adopt a staggering range of phenotypes that are finely tuned responses to a variety of different stimuli, and that the resulting subsets of activated macrophages play critical roles in both progression and resolution of disease. This review summarizes the current understanding of the contributions of differentially polarized macrophages to various infectious and inflammatory diseases and the ongoing effort to develop novel therapies that target this key aspect of macrophage biology.     

Role of Tropomyosin in Formin-mediated Contractile Ring Assembly in Fission Yeast

Like animal cells, fission yeast divides by assembling actin filaments into a contractile ring. In addition to formin Cdc12p and profilin, the single tropomyosin isoform SpTm is required for contractile ring assembly. Cdc12p nucleates actin filaments and remains processively associated with the elongating barbed end while driving the addition of profilin-actin. SpTm is thought to stabilize mature filaments, but it is not known how SpTm localizes to the contractile ring and whether SpTm plays a direct role in Cdc12p-mediated actin polymerization. Using “bulk” and single actin filament assays, we discovered that Cdc12p can recruit SpTm to actin filaments and that SpTm has diverse effects on Cdc12p-mediated actin assembly. On its own, SpTm inhibits actin filament elongation and depolymerization. However, Cdc12p completely overcomes the combined inhibition of actin nucleation and barbed end elongation by profilin and SpTm. Furthermore, SpTm increases the length of Cdc12p-nucleated actin filaments by enhancing the elongation rate twofold and by allowing them to anneal end to end. In contrast, SpTm ultimately turns off Cdc12p-mediated elongation by “trapping” Cdc12p within annealed filaments or by dissociating Cdc12p from the barbed end. Therefore, SpTm makes multiple contributions to contractile ring assembly during and after actin polymerization.     

Essential Role of the Dynamin Pleckstrin Homology Domain in Receptor-Mediated Endocytosis

Pleckstrin homology (PH) domains are found in numerous membrane-associated proteins and have been implicated in the mediation of protein-protein and protein-phospholipid interactions. Dynamin, a GTPase required for clathrin-dependent endocytosis, contains a PH domain which binds to phosphoinositides and participates in the interaction between dynamin and the βγ subunits of heterotrimeric G proteins. The PH domain is essential for expression of phosphoinositide-stimulated GTPase activity of dynamin in vitro, but its involvement in the endocytic process is unknown. We expressed a series of dynamin PH domain mutants in cultured cells and determined their effect on transferrin uptake by those cells. Endocytosis is blocked in cells expressing a PH domain deletion mutant and a point mutant that fails to interact with phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂]. In contrast, expression of a point mutant with unimpaired PI(4,5)P₂ interaction has no effect on transferrin uptake. These results demonstrate the significance of the PH domain for dynamin function and suggest that its role may be to mediate interactions between dynamin and phosphoinositides.     

Microtubules and Microfilaments in Amphibian Neurulation

In the salamander embryo, the morphogenetic movements of neurulationare correlated with two cell shape changes in the neural epithelium:elongation and apical constriction of the columnar neural platecells. Cells first elongate to form the flat open neural plateand then constrict apically as the plate rolls up to form theneural tube. Evidence is presented that these cell shape changesare intrinsic to the cells themselves and that they play a causalrole in the morphogenetic movements. Neural plate cells containnumerous microtubules oriented parallel to the axis of elongation.These microtubules are critical to the elongation process. Possiblemechanisms for microtubule function in cell elongation are considered.During apical constriction the cells contain bundles of microfilamentswhich encircle the cell apex in purse-string fashion. Evidenceis presented which suggests that microfilament bundles playan active role in apical constriction, and that this localizedcontraction is produced by filament sliding.     

A Role for Cdc42 in Macrophage Chemotaxis

Three members of the Rho family, Cdc42, Rac, and Rho are known to regulate the organization of actin-based cytoskeletal structures. In Bac1.2F5 macrophages, we have shown that Rho regulates cell contraction, whereas Rac and Cdc42 regulate the formation of lamellipodia and filopodia, respectively. We have now tested the roles of Cdc42, Rac, and Rho in colony stimulating factor-1 (CSF-1)–induced macrophage migration and chemotaxis using the Dunn chemotaxis chamber. Microinjection of constitutively activated RhoA, Rac1, or Cdc42 inhibited cell migration, presumably because the cells were unable to polarize significantly in response to CSF-1. Both Rho and Rac were required for CSF-1–induced migration, since migration speed was reduced to background levels in cells injected with C3 transferase, an inhibitor of Rho, or with the dominant-negative Rac mutant, N17Rac1. In contrast, cells injected with the dominant-negative Cdc42 mutant, N17Cdc42, were able to migrate but did not polarize in the direction of the gradient, and chemotaxis towards CSF-1 was abolished.

We conclude that Rho and Rac are required for the process of cell migration, whereas Cdc42 is required for cells to respond to a gradient of CSF-1 but is not essential for cell locomotion.

     

In Vivo Role for Actin-regulating Kinases in Endocytosis and Yeast Epsin Phosphorylation

The yeast actin-regulating kinases Ark1p and Prk1p are signaling proteins localized to cortical actin patches, which may be sites of endocytosis. Interactions between the endocytic proteins Pan1p and End3p may be regulated by Prk1p-dependent threonine phosphorylation of Pan1p within the consensus sequence [L/I]xxQxTG. We identified two Prk1p phosphorylation sites within the Pan1p-binding protein Ent1p, a yeast epsin homologue, and demonstrate Prk1p-dependent phosphorylation of both threonines. Converting both threonines to either glutamate or alanine mimics constitutively phosphorylated or dephosphorylated Ent1p, respectively. Synthetic growth defects were observed in a pan1-20 ENT1(EE) double mutant, suggesting that Ent1p phosphorylation negatively regulates the formation/activity of a Pan1p-Ent1p complex. Interestingly, pan1-20 ent2 Delta but not pan1-20 ent1 Delta double mutants had improved growth and endocytosis over the pan1-20 mutant. We found that actin-regulating Ser/Thr kinase (ARK) mutants exhibit endocytic defects and that overexpressing either wild-type or alanine-substituted Ent1p partially suppressed phenotypes associated with loss of ARK kinases, including growth, endocytosis, and actin localization defects. Consistent with synthetic growth defects of pan1-20 ENT1(EE) cells, overexpressing glutamate-substituted Ent1p was deleterious to ARK mutants. Surprisingly, overexpressing the related Ent2p protein could not suppress ARK kinase mutant phenotypes. These results suggest that Ent1p and Ent2p are not completely redundant and may perform opposing functions in endocytosis. These data support the model that, as for clathrin-dependent recycling of synaptic vesicles, yeast endocytic protein phosphorylation inhibits endocytic functions.     

Role of Telomerase in Reactivation of Macrophage Nuclei in Heterokaryons

It was shown that the duration of stay of macrophages in the peritoneal cavity of mice and method of their isolation did not affect markedly their capacity for resumption of DNA synthesis in heterokaryons. This means that mouse macrophage undergo such changes during differentiation that reactivation of DNA synthesis in their nuclei is only possible after interaction of telomeres with telomerase, since it was already shown that telomerase was involved in reactivation of DNA synthesis in the macrophage nuclei. The results of experiments did not reveal differences in the length of telomeres in mouse macrophages and other somatic cells. This could depend on the significant length of mouse telomeres and, as a result, their shortening, sufficient for the inhibition of proliferation, is beyond the limits of sensitivity of the current methods. It is also possible that changes in DNA properties in the macrophages occurring during their differentiation depend on changes in the conformation of the telomere complex in these cells. Testing of this suggestion is relevant with respect to recent data that cell hybridization, specifically in the form of heterokaryons, may be essential in realization of the therapeutic effect caused by the introduction of cells during cell therapy.     

EphA2 Signaling Following Endocytosis: Role of Tiam1

Eph receptors and their membrane‐bound ligands, the ephrins, represent a complex subfamily of receptor tyrosine kinases (RTKs). Eph/ephrin binding can lead to various and opposite cellular behaviors such as adhesion versus repulsion, or cell migration versus cell‐adhesion. Recently, Eph endocytosis has been identified as one of the critical steps responsible for such diversity. Eph receptors, as many RTKs, are rapidly endocytosed following ligand‐mediated activation and traffic through endocytic compartments prior to degradation. However, it is becoming obvious that endocytosis controls signaling in many different manners. Here we showed that activated EphA2 are degraded in the lysosomes and that about 35% of internalized receptors are recycled back to the plasma membrane. Our study is also the first to demonstrate that EphA2 retains the capacity to signal in endosomes. In particular, activated EphA2 interacted with the Rho family GEF Tiam1 in endosomes. This association led to Tiam1 activation, which in turn increased Rac1 activity and facilitated Eph/ephrin endocytosis. Disrupting Tiam1 function with RNA interference impaired both ephrinA1‐dependent Rac1 activation and ephrinA1‐induced EphA2 endocytosis. In summary, our findings shed new light on the regulation of EphA2 endocytosis, intracellular trafficking and signal termination and establish Tiam1 as an important modulator of EphA2 signaling .