The beating of eukaryotic cilia and flagella is controlled by multiple species of inner-arm and outer-arm dyneins. To clarify the regulation on axonemal beating by nucleotide conditions and central-pair microtubules, microtubule sliding in disintegrating Chlamydomonas axonemes of various mutants and in vitro microtubule gliding by isolated axonemal dyneins were examined. In the in vitro motility assays with outer-arm dyneins (alphabeta and gamma), microtubule translocation velocity decreased at high concentrations of ATP, while this inhibition was canceled by the simultaneous presence of ADP or ribose-modified analogues, mantATP/ADP. In contrast, motility of inner-arm dyneins was rather insensitive to these nucleotides. The velocity of sliding disintegration in axonemes lacking the central pair was less than that in wild-type axonemes at high ATP concentrations, but was overcome by the presence of ADP or mantATP/ADP. While these nucleotides did not activate the sliding velocity in other mutant axonemes, they increased the extent of sliding, except for axonemes lacking outer-arm dynein. Experiments with axonemes lacking inner-arm dynein f using casein kinase 1 inhibitor suggest that the regulation of outer-arm dynein by the central pair is effected through the activation of inner-arm dynein f, and possibly by other interactions. These results indicate that the central pair activates outer-arm dyneins on specific outer-doublet, resulting in amplification of the axonemal bending force.
Focal adhesions (FAs) are large clusters of transmembrane receptors of the integrin family and a multitude of associated cytoplasmic "plaque" proteins, which connect the extracellular matrix-bound receptors with the actin cytoskeleton. The formation of nearly stationary FAs defines a boundary between the dense and highly dynamic actin network in lamellipodium and the sparser and more diverse cytoskeletal organization in the lamella proper, creating a template for the organization of the entire actin network. The major "mechanical" and "sensory" functions of FAs; namely, the nucleation and regulation of the contractile, myosin-II-containing stress fibers and the mechanosensing of external surfaces depend, to a major extent, on the dynamics of molecular components within FAs. A central element in FA regulation concerns the positive feedback loop, based on the most intriguing feature of FAs; that is, their dependence on mechanical tension developing by the growing stress fibers. FAs grow in response to such tension, and rapidly disassemble upon its relaxation. In this article, we address the mechanistic relationships between the process of FA development, maturation and dissociation and the dynamic molecular events, which take place in different regions of the FA, primarily in the distal end of this structure (the "toe") and the proximal "heel," and discuss the central role of local mechanical forces in orchestrating the complex interplay between FAs and the actin system.
Angiostatin is a potent inhibitor of angiogenesis. One mechanism through which angiostatin inhibits angiogenesis is by binding to the cell surface protein p80-angiomotin. The p80-angiomotin protein promotes angiogenesis, in part, by conferring a hypermigratory phenotype to endothelial cells. Although p80-angiomotin is extensively characterized, less is known about the related protein angiomotin-like 1. We report that angiomotin-like 1 forms part of a protein complex containing p80-angiomotin. Structure-function studies revealed that angiomotin-like 1 associates with this p80-angiomotin-containing complex via its coiled-coil domain. Since p80-angiomotin plays a role in cell migration, a process that involves the remodeling of the actin cytoskeleton, we then addressed the hypothesis that angiomotin-like 1 may interact with the cytoskeleton. Immunofluorescence studies reveal that angiomotin-like 1 not only co-localizes with filamentous actin but also significantly modifies the architecture of the actin cytoskeleton. Regarding migration, angiomotin-like 1 increases the velocity of migration and decreases the persistence of migration directionality. Together these observations strongly suggest that angiomotin-like 1 is involved in actin-cytoskeleton-based processes, in part, via its interaction with a p80-angiomotin-containing complex and the actin cytoskeleton. These findings have important implications for angiogenesis-driven disease since angiomotin and angiomotin-like 1 are both expressed in capillaries.
The beta-thymosins are N-terminally acetylated peptides of about 5 kDa molecular mass and composed of about 40-44 amino acid residues. The first member of the family, thymosin beta4, was initially isolated from thymosin fraction 5, prepared in five steps from calf thymus. Thymosin beta4 was supposed to be specifically produced and released by the thymic gland and to possess hormonal activities modulating the immune response. Various paracrine effects have indeed been reported for these peptides such as cardiac protection, angiogenesis, stimulation of wound healing, and hair growth. Besides these paracrine effects, it was noted that beta-thymosins occur in high concentration in the cytoplasm of many eukaryotic cells and bind to the cytoskeletal component actin. Subsequently it became apparent from in vitro experiments that they preferentially bind to monomeric (G-)actin and stabilize it in its monomeric form. Due to this ability the beta-thymosins are the main intracellular actin sequestering factor, i.e., they posses the ability to remove monomeric actin from the dynamic assembly and disassembly processes of the actin cytoskeleton that constantly occur in activated cells. In this review we will concentrate on the intracellular activity and localization of the beta-thymosins, i.e., their modulating effect on the actin cytoskeleton.
During a REMI screen for proteins regulating cytokinesis in Dictyostelium discoideum we isolated a mutant forming multinucleate cells. The gene affected in this mutant encoded a kinase, SepA, which is an ortholog of Cdc7, a serine-threonine kinase essential for septum formation in Schizosaccharomyces pombe. Localization of SepA-GFP in live cells and its presence in isolated centrosomes indicated that SepA, like its upstream regulator Spg1, is associated with centrosomes. Knockout mutants of SepA showed a severe cytokinesis defect and a delay in development. In multinucleate SepA-null cells nuclear division proceeded normally and synchronously. However, often cleavage furrows were either missing or atypical: they were extremely asymmetric and constriction was impaired. Cortexillin-I, a marker localizing strictly to the furrow in wild-type cells, demonstrated that large, crescent-shaped furrows expanded and persisted long after the spindle regressed and nuclei returned to the interphase state. Outside the furrow the filamentous actin system of the cell cortex showed strong ruffling activity. These data suggest that SepA is involved in the spatial and temporal control system organizing cortical activities in mitotic and postmitotic cells.
The coordinated formation and release of focal adhesions is necessary for cell attachment and migration. According to current models, these processes are caused by temporal variations in protein composition. Protein incorporation into focal adhesions is believed to be controlled by phosphorylation. Here, we tested the exchange dynamics of GFP-vinculin as marker protein of focal adhesions using the method of Fluorescence Recovery After Photobleaching. The relevance of the phosphorylation state of the protein, the age of focal adhesions and the acting force were investigated. For stable focal adhesions of stationary keratinocytes, we determined an exchangeable vinculin fraction of 52% and a recovery halftime of 57 s. Nascent focal adhesions of moving cells contained a fraction of exchanging vinculin of 70% with a recovery halftime of 36 s. Upon maturation, mean saturation values and recovery halftimes decreased to levels of 49% and 42 s, respectively. Additionally, the fraction of stably incorporated vinculin increased with cell forces and decreased with vinculin phosphorylation within these sites. Experiments on a nonphosphorylatable vinculin mutant construct at phosphorylation site tyr1065 confirmed the direct interplay between phosphorylation and exchange dynamics of adhesion proteins during adhesion site maturation.
The nematode sperm cell crawls by recycling major sperm protein (MSP) from dimers into subfilaments, filaments, and filament complexes, as a result of thermal writhing in the presence of hydrophobic patches. Polymerization near leading edges of the cell intercolates MSP dimers onto the tips of growing filament complexes, forcing them against the cell boundary, and extending the cytoskeleton in the direction of motion. Strong adhesive forces attach the cell to the substrate in the forward part of the lamellipod, while depolymerization in the rearward part of the cell breaks down the cytoskeleton, contracting the lamellipod and pulling the cell body forward. The movement of these cells, then, is caused by coordinated protrusive, adhesive and contractile forces, spatially separated across the lamellipod. This paper considers a phenomenological model that tracks discrete elements of the cytoskeleton in curvilinear coordinates. The pseudo-two dimensional model primarily considers protrusion and rotation of the cell, along with the evolution of the cell boundary. General assumptions are that pH levels within the lamellipod regulate protrusion, contraction and adhesion, and that growth of the cytoskeleton, over time, is perpendicular to the evolving cell boundary. The model follows the growth and contraction of a discrete number of MSP fiber complexes, since they appear to be the principle contributors for force generation in cell boundary protrusion and contraction, and the backbone for the dynamic geometry and motion.
The sea cucumber Holothuria atra is a broadcast spawner. Among broadcast spawners, fertilization occurs by means of an egg-derived substance(s) that induces sperm flagellar motility activation and chemotaxis. Holothuria atra sperm were quiescent in seawater, but exhibited flagellar motility activation near eggs with chorion (intact eggs). In addition, they moved in a helical motion toward intact eggs as well as a capillary filled with the water layer of the egg extracts, suggesting that an egg-derived compound(s) causes motility activation and chemotaxis. Furthermore, demembranated sperm flagella were reactivated in high pH (> 7.8) solution without cAMP, and a phosphorylation assay using (gamma-32P)ATP showed that axonemal protein phosphorylation and dephosphorylation also occurred in a pH-dependent manner. These results suggest that the activation of sperm motility in holothurians is controlled by pH-sensitive changes in axonemal protein phosphorylation. Ca2+ concentration affected the swimming trajectory of demembranated sperm, indicating that Ca2+-binding proteins present at the flagella may be associated with regulation of flagellar waveform. Moreover, the phosphorylation states of several axonemal proteins were Ca2+-sensitive, indicating that Ca2+ impacts both kinase and phosphatase activities. In addition, in vivo sperm protein phosphorylation occurred after treatment with a water-soluble egg extract. Our results suggest that one or more egg-derived compounds activate motility and subsequent chemotactic behavior via Ca2+-sensitive flagellar protein phosphorylation.
Flagellar beating is caused by microtubule sliding, driven by the activity of dynein, between adjacent two of the nine doublet microtubules. An essential process in the regulation of dynein is to alternate its activity (switching) between the two sides of the central pair microtubules. The switching of dynein activity can be detected, in an in vitro system using elastase-treated axonemes of sea urchin sperm flagella, as a reversal of the relative direction of ATP-induced sliding between the two bundles of doublets at high Ca(2+) (10(-4) M) at pH 7.8-8.0. The reversal is triggered by externally applied bending of the doublet bundle. However, the mechanism of this bending-induced reversal (or backward sliding) remains unclear. To understand how the switching of dynein activity in flagella can be induced by bending, we studied the roles of ADP, which is an important factor for the dynein motile activity, and of Ca(2+) in the bending-induced reversal of microtubule sliding between two bundles of doublets at pH 7.5 and 7.2. We found that the reversal of sliding direction was induced regardless of the concentrations of Ca(2+) at low pH, but occurred more frequently at low Ca(2+) (<10(-9) M) than at high Ca(2+). At pH 7.5, an application of ADP increased the frequency of occurrence of backward sliding at high as well as low concentrations of Ca(2+). The results indicate that ADP-dependent activation of dynein, probably resulting from ADP-binding to dynein, is involved in the regulation of the bending-induced switching of dynein activity in flagella.
The use of confocal reflection microscopy (CRM) for the in vivo analysis of microtubule (MT) mediated transport of lipid droplets in the developing Drosophila egg primordia is described here. The developing Drosophila oocytes are ideal objects to study MT-mediated transport in vivo: transport of e.g. the lipid droplets can be conveniently, selectively and sensitively monitored through CRM and the egg primordia are readily available for physical, chemical and/or genetic manipulations. CRM is a non-destructive way to follow vesicle movement and allows high frame rate image recording. When combined with fluorescence imaging, CRM offers simultaneous visualization of the cargo and the protein(s) of interest, i.e. a motor or a cargo adapter, thus allowing a better understanding of MT-mediated transport and spatiotemporal coordination of the transport machinery.
The Wave proteins activate the Arp2/3 complex at the leading edge of migrating cells. The resulting actin polymerization powers the projection of the plasma membrane in lamellipodia and membrane ruffles. The Wave proteins are always found associated with partner proteins. The canonical Wave complex is a stable complex containing five subunits. Even though it is well admitted that this complex plays an essential regulatory role on Wave function, the mechanisms by which Wave proteins are regulated within the complex are still elusive. Even the constitutive activity or inactivity of the complex is controversial. The major difficulty of these assays resides in the long and difficult purification of the Wave complex by a combination of several chromatography steps, which gives an overall low yield and increases the chance of Wave complex denaturation. Here we report a greatly simplified approach to purify the human Wave complex using a stable cell line expressing a tagged subunit and affinity chromatography. This protocol provided us with sufficient amount of pure Wave complex for functional assays. These assays unambiguously established that the Wave complex in its native conformation is intrinsically inactive, indicating that, like WASP proteins, Wave proteins have a masked C-terminal Arp2/3 binding site at resting state. As a consequence, the Wave complex has to be recruited and activated at the plasma membrane to project migration structures. Importantly, the approach we describe here for multiprotein complex purification is likely applicable to a wide range of human multiprotein complexes.
MARK [microtubule-associated proteins (MAPs)/microtubule affinity regulating kinase]/Par-1 (partition defective) phosphorylate MAPs tau, MAP2 and MAP4 at KXGS motifs and thereby regulate microtubule dynamics and transport processes in cells [Drewes et al., Cell1997;89:297-308]. We report here that MARK copurifies with clathrin-coated vesicles (CCVs) via interaction with the adaptor complex AP-2. The adaptin binding site on MARK includes the regulatory loop of its catalytic domain. Immunofluorescence demonstrates the colocalization of MARK with AP-2 and clathrin, as well as other MARK-interacting proteins such as PAK5. The results are consistent with an observed influence of MARK on the trafficking of CCVs. Cell Motil. Cytoskeleton 2009. (c) 2009 Wiley-Liss, Inc.
The Drosophila syncytial embryo uses multiple astral mitotic spindles that are specialized for rapid mitosis. The homotetrameric kinesin-5, KLP61F contributes to various aspects of mitosis in this system, all of which are consistent with it exerting outward forces on spindle poles. In principle, kinesin-5 could accomplish this by (i) sliding microtubules (MTs), minus end leading, relative to a static spindle matrix or (ii) crosslinking and sliding apart adjacent pairs of antiparallel interpolar (ip) MTs. Here, I critically review data on the biochemistry of purified KLP61F, its localization and dynamic properties within spindles, and quantitative modeling of KLP61F function. While a matrix-based mechanism may operate in some systems, the work tends to support the latter "sliding filament" mechanism for KLP61F action in Drosophila embryo spindles. Cell Motil. Cytoskeleton 2009. (c) 2009 Wiley-Liss, Inc.
While matrix stiffness has been implicated in cell adhesion and migration, most studies have focused on the effects of substrate stiffness in 2D. The present work describes a novel continuous stiffness gradient model for studying such processes in 3D. Wedge-shaped collagen scaffolds were compressed to produce sheets of a desired (0.1 mm) uniform thickness, but with increasing collagen density along the length of the sheet. Dynamic mechanical analysis, carried out on 1 mm wide strips obtained from the two ends and the middle of each sheet, showed that the elastic modulus increased from 1057 +/- 487 kPa to 2305 +/- 693 kPa at the soft and stiff end respectively and was 1835 +/- 31 kPa in the middle. In constructs seeded with agarose marker beads prior to compression, mean agarose bead density rose from 10 +/- 1 to 71 +/- 12 at the soft and stiff end respectively and was 19 +/- 5 in the middle, indicating successful engineering of a density gradient corresponding to the measured stiffness gradient. Growth-arrested human dermal fibroblasts, initially seeded evenly within such constructs, accumulated preferentially towards the stiff part of the gradient after 3 and 6 days in culture. Durotactic migration was significant after 6 days. This model provides a new means for studying cellular mechanotaxis and patterning cells which is controllable, biomimetic and in 3D.
The motility, shape, and functionality of the cell depend sensitively on cytoskeletal mechanics which in turn is governed by the properties of filamentous proteins - mainly actin, microtubules, and intermediate filaments. These biopolymers are confined in the dense cytoplasm and therefore experience strong geometric constraints on their equilibrium thermal fluctuations. To obtain a better understanding of the influence of confinement on cytoskeletal filaments we study the thermal fluctuations of individual actin filaments in a microfluidic in vitro system by fluorescence microscopy and determine the persistence length of the filaments by analyzing the radial distribution function. A unique feature of this method is that we obtain the persistence length without detailed knowledge of the complete contour of the filament which makes the technique applicable to a broad range of biological polymers, including those with a persistence length smaller than the optical resolution.
Anisotropic substrata such as micromachined grooves can control cell shape, orientation, and the direction of cell movement, a phenomena termed topographic guidance. Although many types of cells exhibit topographic guidance, little is known regarding cell responses to conflicting topographic cues. We employed a substratum with intersecting grooves in order to present fibroblasts and epithelial cells with conflicting topographic cues. Using time-lapse and confocal microscopy, we examined cell behavior at groove intersections. Migrating fibroblasts and epithelial cells typically extended a cell process into the intersection ahead of the cell body. After travelling along the "X" groove to enter the intersection, the leading lamellipodia of the cell body encountered the perpendicular "Y" groove, and spread latterly along the "Y" groove. The formation of lateral lamellipodia resulted in cells forming "T" or "L" morphologies, which were characterized by the formation of phosphotyrosine-rich focal adhesions at the leading edges. The "Y" groove did not prove an absolute barrier to cell migration, particularly for epithelial cells. Analysis of cytoskeletal distribution revealed that F-actin bundles did not adapt closely to the groove patterns, but typically did align to either the "X" or "Y" grooves. In contrast microtubules (MT) adapted closely to the walls. Inhibition of microtubule nucleation attenuated fibroblast and epithelial cell orientation within the intersection of the perpendicular grooves. We conclude that MT may be the prime determinant of fibroblast and epithelial cell conformation to conflicting topographies.
In previous works we showed that the depolarization of the plasma membrane potential (PMP) determines a reorganization of the cytoskeleton of diverse epithelia in culture, consisting mainly of a reallocation of peripheral actin toward the cell center, ultimately provoking intercellular disruption. In view of this evidence, we explored in this study the possible effects of membrane potential hyperpolarization on the cytoskeletal organization and adherens junction (AJ) morphology and the stability of confluent bovine corneal endothelial cells in culture. For this purpose, hyperpolarization was achieved by substitution of extracellular sodium by nondiffusible cations or via the incorporation of valinomycin to the control solution. Actin compactness at the cell periphery was assessed by quantitative analysis of fluorescence microscopy images. The stability of the AJ was challenged by calcium deprivation or temperature decrease. Our results showed that plasma membrane hyperpolarization provokes a compaction of AJ-associated actin filaments toward the plasma membrane and an increase in the stability of the AJs. We also observed that the hyperpolarizing procedures determined similar modifications in the actin cytoskeleton of endothelial cells in whole bovine corneas. Together with our previous work, the results of this study contribute to the idea that modifications in the PMP of nonexcitable cells participate in cellular adaptive responses involving reorganization of cytoskeletal components.
Metastatic mammary carcinoma cells, which have previously been observed to form mature, matrix degrading invadopodia on a thick ECM matrix, are able to form invadopodia with similar characteristics on glass without previously applied matrix. They form in response to epidermal growth factor (EGF), and contain the usual invadopodium core proteins N-WASP, Arp2/3, cortactin, cofilin, and F-actin. The study of invadopodia on glass allows for higher resolution analysis including the use of total internal reflection microscopy and analysis of their relationship to other cell motility events, in particular, lamellipodium extension and chemotaxis toward an EGF gradient. Invadopodium formation on glass requires N-WASP and cortactin but not microtubules. In a gradient of EGF more invadopodia form on the side of the cells facing the source of EGF. In addition, depletion of N-WASP or cortactin, which blocks invadopodium fromation, inhibits chemotaxis of cells towards EGF. This appears to be a localized defect in chemotaxis since depletion of N-WASP or cortactin via siRNA had no effect on lamellipodium protrusion or barbed end generation at the lamellipodium's leading edge. Since chemotaxis to EGF by breast tumor cells is involved in metastasis, inhibiting N-WASP activity in breast tumor cells might prevent metastasis of tumor cells while not affecting chemotaxis-dependent innate immunity which depends on WASp function in macrophages.
Neutrophils are one type of migrating cell in the body's innate immune system and are the first line of defense against inflammation or infection. While extensive work exists on the effect of adhesive proteins on neutrophil motility, little is known about how neutrophil motility is affected by the mechanical properties of their physical environment. This study investigated the effects of substrate stiffness on the morphology, random motility coefficient, track speed (v), spreading area, and distribution of turning angles of neutrophils during chemokinesis. Human neutrophils were plated onto polyacrylamide gels of varying stiffness, ranging from 3 to 13 kPa, and coated with the extracellular matrix protein fibronectin, and timelapse images were taken with phase contrast microscopy. Our results show a biphasic behavior between neutrophil motility and substrate stiffness, with the optimum stiffness for motility depending on the concentration of fibronectin on the surface of the gel. On 100 microg/mL fibronectin, the optimum stiffness is 4 kPa (v = 6.9 +/- 0.6 microm/min) while on 10 microg/mL fibronectin, the optimum stiffness increases to 7 kPa (v = 4.5 +/- 2.0 microm/min). This biphasic behavior most likely arises because neutrophils on soft gels are less adherent, preventing production of traction forces, while neutrophils on stiff gels adhere strongly, resulting in decreased migration. At intermediate stiffness, however, neutrophils can attain optimal motility as a function of extracellular matrix coating.
The Cilium, the Nucleus and the Mitochondrion are three important organelles whose evolutionary histories are intimately related to the evolution and origin of the eukaryotic cell. The cilium is involved in motility and sensory transduction. The cilium is only found in the eukaryotic cells. Here we show that eight gene duplications prior to the last common ancestor of all extant eukaryotes account for the expansion of the Heavy Chain Dynein family of motor proteins and the evolution of the complexity of the cilium. The ambiguities in the branching of the phylogenetic tree of the HC-Dyneins were resolved by creating well-defined subtrees and using them to create the full tree. Due to the intimate relationship between the nucleus, the division center, mitosis and the basal body/centriole, the evolution of the cilium can now be related to the evolution of mitosis. In addition, the analysis of the cilium rules out its endosymbiotic origin from a phagocytosis of a bacterium.
