Centripetal transport of cytoplasm, actin, and the cell surface in lamellipodia of fibroblasts

Wound healing in Swiss 3T3 cultures was investigated with video-enhanced contrast (VEC) microscopy. The formation of protrusions at the leading edge of cells along wound was investigated in detail during the spreading stage, which usually lasted from 1 to 4 hr postwounding. Lamellipodia exhibited a continuous rearward, or centripetal, transport of a variety of cellular constituents at rates of approximately 0.26 microns/sec from the leading edge. The lamellipodia were also the sites of lateral migration as well as extension and retraction of actin microspikes. Actin fibers oriented transversely to the direction of movement were also observed to transport centripetally at similar rates. These fibers may in part give rise to large actin fibers forming at the interface between the base of the lamellipodia and the lamellae. Beads 0.5 microns in diameter attached to the dorsal surfaces of lamellipodia also transported centripetally at rates of approximately 0.21 microns/sec. Thus there is an apparent correlation between transport of a variety of structures within lamellipodia and with surface movements of lamellipodia.     

Imaging native beta-actin mRNA in motile fibroblasts

Nuclease-resistant, cytoplasmically resident molecular beacons were used to specifically label beta-actin mRNA in living and motile chicken embryonic fibroblasts. beta-actin mRNA signals were most abundant in active lamellipodia, which are protrusions that cells extend to adhere to surfaces. Time-lapse images show that the immediate sources of beta-actin mRNA for nascent lamellipodia are adjacent older protrusions. During the development of this method, we observed that conventional molecular beacons are rapidly sequestered in cell nuclei, leaving little time for them to find and bind to their cytoplasmic mRNA targets. By linking molecular beacons to a protein that tends to stay within the cytoplasm, nuclear sequestration was prevented, enabling cytoplasmic mRNAs to be detected and imaged. Probing beta-actin mRNA with these cytoplasmically resident molecular beacons did not affect the motility of the fibroblasts. Furthermore, mRNAs bound to these probes undergo translation within the cell. The use of cytoplasmically resident molecular beacons will enable further studies of the mechanism of beta-actin mRNA localization, and will be useful for understanding the dynamics of mRNA distribution in other living cells.     

Dynamic actin patterns and Arp2/3 assembly at the substrate-attached surface of motile cells

BACKGROUND: In the cortical region of motile cells, the actin network rapidly reorganizes as required for movement in various directions and for cell-to-substrate adhesion. The analysis of actin network dynamics requires the combination of high-resolution imaging with a specific fluorescent probe that highlights the filamentous actin structures in live cells. RESULTS: Combining total internal reflection fluorescence (TIRF) microscopy with a method for labeling actin filaments, we analyze the dynamics of actin patterns in the highly motile cells of Dictyostelium. A rapidly restructured network of single or bundled actin filaments provides a scaffold for the assembly of differentiated actin complexes. Recruitment of the Arp2/3 complex characterizes stationary foci with a lifetime of 7-10 s and traveling waves. These structures are also formed in the absence of myosin-II. Arp2/3-actin assemblies similar to those driving the protrusion of a leading edge form freely at the inner face of the plasma membrane. CONCLUSIONS: The actin system of highly motile cells runs far from equilibrium and generates a multitude of patterns within a dynamic filamentous network. Traveling waves are the most complicated patterns based on recruitment of the Arp2/3 complex. They are governed by the propagated induction of actin polymerization. We hypothesize that the actin system autonomously generates primordia of specialized structures such as phagocytic cups or lamellipodia. These primordia would represent an activated state of the actin system and enable cells to respond within seconds to local stimuli by chemotaxis or phagocytic-cup formation.     

Presenilin 1 expression on the cell surface in motile polarized cells

Most cases of early-onset familial Alzheimer’s disease are caused by mutations in the presenilin 1 gene. Nonetheless, the function of presenilin 1 are not yet completely understood. It was shown that endogenous presenilin 1, as well as the adhesion protein CD44, is concentrated on the surface of lamellipodia of polarized T cells (Jurkat cells) after adhesion to a collagen matrix. This phenomenon was not observed for another surface protein of T cells, T cell receptor, which is not involved in cell adhesion processes. In cultures of primary mouse cortical neurons, presenilin 1 was concentrated on the surface of the growth cone and at neurite contact sites. The concentration of presenilin 1 on the surface of structures that determine cell motility and intercellular contacts suggests that presenilin 1 plays an important role in cell adhesion in motile polarized cells.     

Expression of presenilin 1 on the cell surface in motile polarized cells

Most cases of familial early-onset Alzheimer's disease are caused by mutations in the presenilin 1 (PS1) gene. However, the cellular functions of PS1 are not yet completely understood. We showed that endogenous PS1 and the adhesion protein CD44 are redistributed on the surface of cell projections (lamellipodia) in polarized T- lymphocytes (Jurkat cells) after the adhesion to a collagen matrix. This effect was not observed for another surface protein of T lymphocytes, which is not involved in cell adhesion processes, the T cell receptor. In primary cultures of mouse cortical neurons, PS1 was concentrated at the surface of extended growth cones and at the sites of neurite contacts. The concentration of PS1 at the surface of cellular structures that promote cell motility and cell contacts suggests an important role of PSI in cell adhesion in motile polarized cells.     

Interior decoration: Tropomyosin in actin dynamics and cell migration

Cell migration and invasion requires the precise temporal and spatial orchestration of a variety of biological processes. Filaments of polymerized actin are critical players in these diverse processes, including the regulation of cell anchorage points (both cell-cell and cell-extracellular matrix), the uptake and delivery of molecules via endocytic pathways and the generation of force for both membrane protrusion and retraction. How the actin filaments are specialized for each of these discrete functions is yet to be comprehensively elucidated. The cytoskeletal tropomyosins are a family of actin associating proteins that form head-to-tail polymers which lay in the major groove of polymerized actin filaments. In the present review we summarize the emerging isoform-specific functions of tropomyosins in cell migration and invasion and discuss their potential roles in the specialization of actin filaments for the diverse cellular processes that together regulate cell migration and invasion.Key words: tropomyosin, actin, migration, invasion, cytoskeleton, actin dynamics, adhesionActin is the most abundant protein in eukaryotic cells and is critical for maintaining structural integrity. The polymerization of globular (G)-actin monomers forms actin filaments (F-actin),¹ which play a role in diverse and complex cellular functions including intercellular transport of organelles and vesicles,^2,3 cytokinesis,⁴ apoptosis⁵ and cell motility.⁶ Intricate details describing the molecular scale interactions between regulatory proteins and actin have been extensively investigated but the mechanistic control of diverse actin filament functions remain largely unclear. Recent improvements in analysis techniques⁷ and the use of physiologically relevant models of 3D cell culturing⁸ have now begun to reveal mechanisms of actin cytoskeleton regulation. Accruing evidence suggests that the actin decorating protein tropomyosin is a key regulator of actin filament specialization. Of particular interest is the impact that tropomyosin regulation has on actin filament activity during cell migration and invasion that underpins immunological cell homing, development, wound healing and metastasis.     

Reduced cell migration and disruption of the actin cytoskeleton in calpain-deficient embryonic fibroblasts

The physiological functions and substrates of the calcium-dependent protease calpain remain only partly understood. The mu- and m-calpains consist of a mu- or m-80-kDa large subunit (genes Capn1 and Capn2), and a common 28-kDa small subunit (Capn4). To assess the role of calpain in migration, we used fibroblasts obtained from Capn4(-/-) mouse embryos. The cells lacked calpain activity on casein zymography and did not generate the characteristic calpain-generated spectrin breakdown product that is observed in wild-type cells. Capn4(-/-) cells had decreased migration rates and abnormal organization of the actin cytoskeleton with a loss of central stress fibers. Interestingly, these cells extended numerous thin projections and displayed delayed retraction of membrane protrusions and filopodia. The number of focal adhesions was decreased in Capn4(-/-) cells, but the cells had prominent vinculin-containing focal complexes at the cell periphery. The levels of the focal adhesion proteins, alpha-actinin, focal adhesion kinase (FAK), spectrin, talin, and vinculin, were the same in Capn4(+/+) and Capn4(-/-) cells. FAK, alpha-actinin, and vinculin were not cleaved in either cell type plated on fibronectin. However, proteolysis of the focal complex component, talin, was detected in the wild-type cells but not in the Capn4(-/-) cells, suggesting that calpain cleavage of talin is important during cell migration. Moreover, talin cleavage was again observed when calpain activity was partially restored in Capn4(-/-) embryonic fibroblasts by stable transfection with a vector expressing the rat 28-kDa calpain small subunit. The results demonstrate unequivocally that calpain is a critical regulator of cell migration and of the organization of the actin cytoskeleton and focal adhesions.     

The nucleation-release model of actin filament dynamics in cell motility

The actin cytoskeleton is intimately involved in the motile behaviour of animal cells. The structure and dynamic behaviour of actin and its binding proteins have been intensively studied in vitro over the past several decades, culminating in achievements such as an atomic model of the actin filament. Despite this progress, it is not yet clear how the behaviour of these purified proteins in vitro relates to the dynamic behaviour of actin inside living, moving cells. Here we discuss a new model that relates the known dynamic parameters for pure actin to the observed behaviour of actin filaments inside motile cells.     

Clasps are CLIP-115 and -170 associating proteins involved in the regional regulation of microtubule dynamics in motile fibroblasts

CLIP-170 and CLIP-115 are cytoplasmic linker proteins that associate specifically with the ends of growing microtubules and may act as anti-catastrophe factors. Here, we have isolated two CLIP-associated proteins (CLASPs), which are homologous to the Drosophila Orbit/Mast microtubule-associated protein. CLASPs bind CLIPs and microtubules, colocalize with the CLIPs at microtubule distal ends, and have microtubule-stabilizing effects in transfected cells. After serum induction, CLASPs relocalize to distal segments of microtubules at the leading edge of motile fibroblasts. We provide evidence that this asymmetric CLASP distribution is mediated by PI3-kinase and GSK-3 beta. Antibody injections suggest that CLASP2 is required for the orientation of stabilized microtubules toward the leading edge. We propose that CLASPs are involved in the local regulation of microtubule dynamics in response to positional cues.     

Density and cell cycle dependence of cell surface proteins in hamster fibroblasts

The large, external, transformation-sensitive (LETS) glycoprotein of hamster fibroblasts, detected by lactoperoxidase-catalyzed iodination, is shown to vary in its accessibility at the cell surface, depending upon the growth state and position in the cell cycle. High levels correlate with arrest in early G1, and these fall after growth stimulation by serum. Very low levels are detectable on mitotic cells.     

Mathematical modelling and numerical simulations of actin dynamics in the eukaryotic cell

The aim of this article is to study cell deformation and cell movement by considering both the mechanical and biochemical properties of the cortical network of actin filaments and its concentration. Actin is a polymer that can exist either in filamentous form (F-actin) or in monometric form (G-actin) (Chen et al. in Trends Biochem Sci 25:19–23, 2000) and the filamentous form is arranged in a paired helix of two protofilaments (Ananthakrishnan et al. in Recent Res Devel Biophys 5:39–69, 2006). By assuming that cell deformations are a result of the cortical actin dynamics in the cell cytoskeleton, we consider a continuum mathematical model that couples the mechanics of the network of actin filaments with its bio-chemical dynamics. Numerical treatment of the model is carried out using the moving grid finite element method (Madzvamuse et al. in J Comput Phys 190:478–500, 2003). Furthermore, by assuming slow deformations of the cell, we use linear stability theory to validate the numerical simulation results close to bifurcation points. Far from bifurcation points, we show that the mathematical model is able to describe the complex cell deformations typically observed in experimental results. Our numerical results illustrate cell expansion, cell contraction, cell translation and cell relocation as well as cell protrusions. In all these results, the contractile tonicity formed by the association of actin filaments to the myosin II motor proteins is identified as a key bifurcation parameter.     

Molecular dynamics and forces of a motile cell simultaneously visualized by TIRF and force microscopies

Cells must exert traction forces onto the substratum for continuous migration. Molecular dynamics such as actin polymerization at the front of the cell and myosin II accumulation at the rear should play important roles in the exertion of forces required for migration. Therefore, it is important to reveal the relationships between the traction forces and molecular dynamics. Traction forces can be calculated from the deformation of the elastic substratum under a migrating cell. A transparent and colorless elastic substratum with a high refractive index (1.40) and a low Young's modulus (1.0 kPa) were made from a pair of platinum-catalyzed silicones. We used this substratum to develop a new method for simultaneous recording of molecular dynamics and traction forces under a migrating cell in which total internal refractive fluorescence (TIRF) and force microscopies were combined. This new method allows the detection of the spatiotemporal distribution of traction forces produced by individual filopodia in migrating Dictyostelium cells, as well as simultaneous visualization of these traction forces and the dynamics of filamentous myosin II.     

Sequestered actin in chick embryo fibroblasts

Chick embryo fibroblasts contain about 75-100 M unpolymerized actin and at least four proteins which can bind actin monomers, actin depolymerizing factor (ADF), gelsolin, profilin, and thymosin ₄ (T₄). Fibroblast extracts are analyzed by non-denaturing polyacrylamide gel electrophoresis and immunoblotting where most of the G-actin is detected as a complex with T₄. When fibroblast extracts are fractionated by gel filtration and the fractions are analyzed by PAGE and HPLC, most of the G-actin elutes in a peak that also contains T₄ at an overall molar ratio of 1.9:1 relative to actin. Gelsolin, profilin, and ADF are also detectable in the gel filtration eluate and at least partly coelute with actin, and account for only a minor fraction of the soluble actin pool. These observations indicate that under the growth conditions studied, T₄ is the major actin-sequestering protein in fibroblasts.     

Concanavalin A surface receptors and cytoplasmic actin in cell adhesion.

A double immunofluorescence staining technique to locate concanavalin A (Con A) surface receptors and cytoplasmic actin in the same cell was applied to monolayer cultures of rat foetal fibroblasts during cell detachment induced by trypsin and during cell attachment to glass substratum. Con A receptors were demonstrated by fluorescein-isothiocyanate-labelled Con A (FITC-Con A) and actin by specific anti-actin antibody (AAA) traced with rhodamine-labelled goat anti-human globulin (R-AHG). Untreated, control cells had an elongated shape, Con A receptors restricted to cell margins and prominent actin filaments. After 2 min treatment with 0.001% trypsin the cells became angular with Con A receptors in clusters and actin in a diffuse or aggreagate staining pattern. Progressive cell rounding followed and this was accompanied by the development of long, thin, arborized cell processes, studded with Con A receptors and containing fine actin filaments. Complete cell rounding preceded cell detachment. The sites of detached cells were marked by fine aggregates containing Con A receptors and actin. In cells attaching to a glass substratum, actin was present in a diffusely stained or aggregate pattern in round cells, in filaments restricted to cell margins in partially spread cells and in numerous filaments in fully spread cells. Con A receptors were present in clusters in round cells, in clusters or caps in partially spread cells and in cell margins in fully spread cells. Binding of FITC-Con A to partially spread cells resulted in dissolution of the few, newly formed, actin filaments. We believe our observations are consistent with the idea that actin filaments, formed during cell attachment, contribute towards the maintenance of cell adhesion by helping in the preservation of cell shape and by anchorage of Con A receptors at points of cell attachment to the substratum.     

Cell adhesion dynamics and actin cytoskeleton reorganization in HepG2 cell aggregates

The temporal dependence of cytoskeletal remodelling on cell-cell contact in HepG2 cells has been established here. Cell-cell contact occurred in an ultrasound standing wave trap designed to form and levitate a 2-D cell aggregate, allowing intercellular adhesive interactions to proceed, free from the influences of solid substrata. Membrane spreading at the point of contact and change in cell circularity reached 50% of their final values within 2.2 min of contact. Junctional F-actin increased at the interface but lagged behind membrane spreading, reaching 50% of its final value in 4.4 min. Aggregates had good mechanical stability after 15 min in the trap. The implication of this temporal dependence on the sequential progress of adhesion processes is discussed. These results provide insight into how biomimetic cell aggregates with some liver cell functions might be assembled in a systematic, controlled manner in a 3-D ultrasound trap.