Forms Of Cell Motility Research Articles

Integrative experimental/computational approach establishes active cellular protrusion as the primary driving force of phagocytic spreading by immune cells.

The dynamic interplay between cell adhesion and protrusion is a critical determinant of many forms of cell motility. When modeling cell spreading on adhesive surfaces, traditional mathematical treatments often consider passive cell adhesion as the primary, if not exclusive, mechanistic driving force of this cellular motion. To better assess the contribution of active cytoskeletal protrusion to immune-cell spreading during phagocytosis, we here develop a computational framework that allows us to optionally investigate both purely adhesive spreading (“Brownian zipper hypothesis”) as well as protrusion-dominated spreading (“protrusive zipper hypothesis”). We model the cell as an axisymmetric body of highly viscous fluid surrounded by a cortex with uniform surface tension and incorporate as potential driving forces of cell spreading an attractive stress due to receptor-ligand binding and an outward normal stress representing cytoskeletal protrusion, both acting on the cell boundary. We leverage various model predictions against the results of a directly related experimental companion study of human neutrophil phagocytic spreading on substrates coated with different densities of antibodies. We find that the concept of adhesion-driven spreading is incompatible with experimental results such as the independence of the cell-spreading speed on the density of immobilized antibodies. In contrast, the protrusive zipper model agrees well with experimental findings and, when adapted to simulate cell spreading on discrete adhesion sites, it also reproduces the observed positive correlation between antibody density and maximum cell-substrate contact area. Together, our integrative experimental/computational approach shows that phagocytic spreading is driven by cellular protrusion, and that the extent of spreading is limited by the density of adhesion sites.

First person – Robert Bloodgood and Roger Sloboda

ABSTRACT First Person is a series of interviews with the authors of a selection of papers published in Journal of Cell Science. Here, established researchers Robert Bloodgood and Roger Sloboda, authors on ‘The Chlamydomonas flagellar membrane glycoprotein FMG-1B is necessary for expression of force at the flagellar surface’, published in JCS, talk about their paper and career. Robert is a Professor at Department of Cell Biology, University of Virginia School of Medicine, VA, and Roger is a Professor at Department of Biological Sciences, Life Science Center, Dartmouth College, NH. They are interested in understanding the mechanism of an unusual form of cell motility, flagellar-dependent whole-cell gliding in the bi-flagellate green alga Chlamydomonas.

Traction Stress Dynamics During Chemotactic Amoeboid Cell Migration

Chemotaxis is involved in a broad range of biological phenomena such as during cancer metastasis. It requires a tightly regulated, spatiotemporal coordination of underlying biochemical processes that impact the mechanics of cell migration. In response to intrinsic and environmental cues, motile cells can adapt their migration effectively. Yet both the mechanisms by which this adaptation occurs and the role of the interactions between biochemistry and mechanics of cell migration are largely unknown.We use Fourier Traction Force Microscopy to measure the spatiotemporal evolution of shape and traction stresses and construct traction tension kymographs to analyze cell motility as a function of the dynamics of the cells' mechanically active adhesions (traction adhesions). We show that wild-type cells migrate mainly by forming two stationary traction adhesion sites at their front and back halves, over which the cell body moves forward in a step-wise fashion through periodic axial and, to a lesser degree, lateral contractions. We demonstrate that lateral forces are critical in mediating cell motility and essential for migration on highly adhesive substrates where cells implement two alternate motility modes to achieve migration. Our analysis of two mutant strains that lack distinct F-actin crosslinkers (mhcA- and abp120- cells) also supports a key role for lateral contractions in amoeboid cell motility, while the differences in their traction adhesion dynamics suggest the two mutant strains use distinct mechanisms to achieve migration. The considerable differences we find in both the spatiotemporal organization of traction adhesions and contractility, when comparing to the control wild type, provide insight into the role of the extracellular environment and of key cytoskeletal proteins in cell migration. We propose that these are highly conserved mechanisms, which function in a range of amoeboid cells, including leukocytes, as well as other forms of cell motility.

Amoeboid Cells Migrate by Alternating Between Modes with Distinct Adhesion Dynamics and Contractility

Directional cell migration is involved in a broad range of biological phenomena, ranging from the metastatic spreading of cancer to wound healing. Chemotaxing Dictyostelium cells adapt their morphology and speed to external conditions like the stiffness and adhesive properties of their substrate. The mechanism by which they control both their shape and speed remains largely unknown. Using Traction Force Microscopy measurements, we construct traction tension kymographs to examine the spatio-temporal dynamics of both the adhesions and the traction stresses during migration. We show that wild-type cells control their motility by switching between two motility modes with distinct adhesion and contractility dynamics. In the “Stepping-Stepping” mode, the adhesion sites remain stationary while the cell moves forward by periodic axial contractions. The back adhesions break after new frontal adhesions are formed. In the “Stepping-Gliding” mode, the cell reduces the magnitude of the traction stresses, increases the frequency of axial contractions and its migration speed, and keeps the frontal adhesion stationary while sliding the back adhesion forward. These two modes are not conserved when cells move on adhesive poly-L-Lys coated substrates, where cells alternate between a “Nearly Stationary” mode, characterized by strong lateral contractions and extremely low migration speed and a “Gliding-Gliding” mode, where multiple weak and transient adhesions are formed which are gliding forward as the cell moves by barely adhering to the substrate. In summary, our findings have contributed to a more precise understanding of how the coordination of traction stresses together with the adhesion dynamics result in efficient amoeboid cell migration. We propose that these are highly conserved mechanisms, which function in a range of amoeboid cells, including leukocytes, as well as other forms of cell motility.

Regulation of Actin Isoforms by Nitric Oxide

Nitric oxide is a known regulator of cellular function that can act indirectly via activation of guanylyl cyclase, or directly via the S-nitrosylation of cysteine residues. However, little is known about the breadth of proteins that are regulated. Actin is present in nearly all eukaryotic cells and in a variety of isoforms that differ slightly in amino acid sequence, and more specifically in the number and placement of cysteine residues. It has been shown by others that different cysteines in the various isoforms can be modified by a number of oxidation reactions including S-nitrosylation. We therefore sought to determine the effect of S-nitrosylation on the interactions of skeletal, smooth, and non-muscle actins with myosin. We measured skeletal, smooth, and non-muscle (β and γ) actin nitrosylation in response to in vitro treatment with nitroso-L-cysteine - an endogenous nitrosothiol and NO donor - as well as actin filament velocity over heavy meromyosin (HMM), and the actin activated ATPase rates of HMM. A coumarin switch assay showed that all three isoforms could be nitrosylated by nitroso-L-cysteine. Nitrosylation of each actin isoform significantly decreased the in vitro velocity over HMM by approximately 38% with 50 μM donor compared to untreated controls. In contrast, there was no difference observed in the α-skeletal actin activated ATPase rates of the control and treated filaments. Further studies are being conducted to determine which cysteine residues in skeletal, smooth, and non-muscle actin are modified by nitroso-L-cysteine. Together these data suggest that nitrosylation of actin affects the attached time of actomyosin crossbridges. Given the ubiquitous nature of actin it is possible that nitrosylation is a common regulatory scheme for muscle contraction and various forms of cell motility.

Peptidase-resistant peptide inhibitors of myosin light chain kinase

Myosin light chain kinase (MLCK) is the key regulator of various forms of cell motility including endothelial and epithelial permeability in particular. One of the potential MLCK inhibitors to be used in humans is a membrane permeable peptide H-RKKYKYRRK-NH2 (L-PIK). In present work we used solid phase peptide synthesis and Fmoc-technology to produce five modifications of L-PIK. Based on (1)H NMR analysis revealed that these peptides demonstrated improved resistance to degradation in blood plasma. One of de novo synthesized peptides, L-[MeArg(1)]PIK inhibited MLCK activity in vitro with the same efficiency as L-PIK whereas other modified peptides showed reduced inhibitory activity. D-amino acid analog of PIK was the least active inhibitor. Thus, we have demonstrated the possibility to produce an effective MLCK peptide inhibitor with increased resistance to biodegradation that is suitable for further pharmacological development.

Cortactin is necessary for E-cadherin-mediated contact formation and actin reorganization.

Classical cadherin adhesion molecules are key determinants of cell–cell recognition during development and in post-embryonic life. A decisive step in productive cadherin-based recognition is the conversion of nascent adhesions into stable zones of contact. It is increasingly clear that such contact zone extension entails active cooperation between cadherin adhesion and the force-generating capacity of the actin cytoskeleton. Cortactin has recently emerged as an important regulator of actin dynamics in several forms of cell motility. We now report that cortactin is recruited to cell–cell adhesive contacts in response to homophilic cadherin ligation. Notably, cortactin accumulates preferentially, with Arp2/3, at cell margins where adhesive contacts are being extended. Recruitment of cortactin is accompanied by a ligation-dependent biochemical interaction between cortactin and the cadherin adhesive complex. Inhibition of cortactin activity in cells blocked Arp2/3-dependent actin assembly at cadherin adhesive contacts, significantly reduced cadherin adhesive contact zone extension, and perturbed both cell morphology and junctional accumulation of cadherins in polarized epithelia. Together, our findings identify a necessary role for cortactin in the cadherin–actin cooperation that supports productive contact formation.

Actin crystal dynamics: structural implications for F-actin nucleation, polymerization, and branching mediated by the anti-parallel dimer

Actin filament nucleation, polymerization, and branching are crucial steps in many forms of cell motility, cell shape, and intracellular organelle movements in a wide range of organisms. Previous biochemical data suggests that an anti-parallel actin dimer can incorporate itself into growing filamentous actin (F-actin) and has a role in branching. Furthermore, it is a widespread belief that nucleation is spawned from an actin trimer complex. Here we present the structures of actin dimers and trimers in two tetragonal crystal systems P4 32 12 and P4 3. Both crystal systems formed by an induced condensation transformation of a previously reported orthorhombic crystal system P2 12 12 1. Comparison between the three crystal systems demonstrates the dynamics and flexibility of actin–actin interactions. The dimer and trimer actin rearrangements observed between the three crystal systems may provide insight to in vivo actin–actin interactions that occur during the nucleation, polymerization, and branching of F-actin.

Vaccinia virus induces Ca2+-independent cell-matrix adhesion during the motile phase of infection.

Vaccinia virus (VV) induces two forms of cell motility: cell migration, which is dependent on the expression of early genes, and the formation of cellular projections, which requires the expression of late genes. The need for viral gene expression prior to cell motility suggests that VV proteins may affect how infected cells interact with the extracellular matrix. To address this, we have analyzed changes in cell-matrix adhesion after infection of BS-C-1 cells with VV. Whereas uninfected cells round up and detach from the culture flask in the presence of EGTA, infected cells remain attached to the culture flask with a stellate morphology. Ca2+-independent cell-matrix adhesion was evident by 10 h postinfection, after the onset of cell motility but before the formation of virus-induced cellular projections. Progression to Ca2+-independent adhesion required the expression of late viral genes but not the formation of intracellular enveloped virus particles or intracellular actin tails. Analyses of specific matrix proteins identified vitronectin and fibronectin as optimal ligands for Ca2+-independent adhesion and the formation of cellular projections. Adhesion to fibronectin was mediated via RGD motifs alone and was not inhibited by 500 micrograms of heparin/ml. Kistrin, a disintegrin which binds preferentially to the alphav beta3 (vitronectin/fibronectin) receptor inhibited the formation of cellular projections without disrupting preformed matrix interactions. Finally, we show that Ca2+-independent cell-matrix adhesion is a dynamic process which mediates changes in the morphology of VV-infected cells and uninfected cells which exhibit a transformed phenotype.

Motility behavior of hepatocytes on extracellular matrix substrata during aggregation

Aggregation of hepatocytes in culture is an important phenomenon to control in tissue engineering applications. Aggregation generally enhances maintenance of differentiated functions but inhibits cell growth. At present there exists insufficient information for rational design of substrata that control aggregation. Indeed, the cellular mechanism(s) underlying the aggregation process is poorly understood, although cell motility is generally considered to be an essential phenomenon. In this article we provide the first study investigating the relationship between hepatocyte aggregation and motility behavior on various extracellular matrix substrata, including Matrigel, laminin, and fibronectin. We find that the extent of aggregation depends on the concentration of the extracellular matrix proteins, as well as on the type. Furthermore, we find that the extent of aggregation appears to be independent of classical single-cell locomotion. In fact, under conditions giving rise to substantial aggregation, the fraction of cells exhibiting classical locomotion is essentially negligible. Instead, aggregation appears to involve intracellular contacts accomplished via a different form of cell motility: active cell membrane extensions followed by adhesive cell–cell interactions. An implication of these findings is that aggregation may be largely governed by relative strengths of cell–cell versus cell–substratum interactions. These observations could be helpful for improved design of cell transplantation devices and cell culture substrata. © 1996 by John Wiley & Sons, Inc.

Motility behavior of hepatocytes on extracellular matrix substrata during aggregation

Aggregation of hepatocytes in culture is an important phenomenon to control in tissue engineering applications. Aggregation generally enhances maintenance of differentiated functions but inhibits cell growth. At present there exists insufficient information for rational design of substrata that control aggregation. Indeed, the cellular mechanism(s) underlying the aggregation process is poorly understood, although cell motility is generally considered to be an essential phenomenon. In this article we provide the first study investigating the relationship between hepatocyte aggregation and motility behavior on various extracellular matrix substrata, including Matrigel, laminin, and fibronectin. We find that the extent of aggregation depends on the concentration of the extracellular matrix proteins, as well as on the type. Furthermore, we find that the extent of aggregation appears to be independent of classical single-cell locomotion. In fact, under conditions giving rise to substantial aggregation, the fraction of cells exhibiting classical locomotion is essentially negligible. Instead, aggregation appears to involve intracellular contacts accomplished via a different form of cell motility: active cell membrane extensions followed by adhesive cell-cell interactions. An implication of these findings is that aggregation may be largely governed by relative strengths of cell-cell versus cell-substratum interactions. These observations could be helpful for improved design of cell transplantation devices and cell culture substrata. (c) 1996 by John Wiley & Sons, Inc.

Actin in cochlear outer hair cells: A structural basis for cell shape and motility

Threshold sensitivity and frequency selectivity in the cochlea depend on intact outer hair cells and models to explain these phenomena suggest that active mechanical processes, residing in outer hair cells, alter micromechanical properties of the basilar membrane. Auditory sensory hair cells isolated from the inner ear change their shape in response to stimulation in vitro. Three areas of the cell have been suggested to be involved in this form of cell motility: (1) the infracuticular network of actin filaments that is seen as a central core; (2) the cortical layer of actin filaments along the lateral wall; and (3) actin in the outer hair cell cytoplasm. The actin filaments in the stereocilia, the cuticular plate, and the infracuticular network are not involved, since mechanically disrupted cells lacking these structures still shorten. Actin filaments along the lateral wall are not involved in shortening since outer hair cell membrane ghosts do not shorten, however, they retain the ability to constrict so...

On the mechanism of a high-frequency force generator in outer hair cells isolated from the guinea pig cochlea.

Isolated mammalian outer hair cells elongate or shorten respectively by several micrometres when electrically hyperpolarized or depolarized. The experiments in this paper were designed to locate the force-generating mechanism that drives length changes in outer hair cells, and to determine some of its basic properties. The whole-cell mode of the patch-clamp technique was used to stimulate cells electrically and to perfuse them with specific drugs. The pattern of displacement of cellular organelles, and the relative displacements of the cell base and apex during electrical stimulation with the cell mechanically anchored at various points along its length, suggest that the force-generating mechanism is distributed throughout the length of the cell. Further experiments altering the shape, volume and intracellular pressure of outer hair cells suggest that the mechanism is closely associated with the plasma membrane. These experiments also demonstrate that the characteristic tubular shape of outer hair cells is maintained by membrane-associated structures with elastic properties that enable the cell to return to its original shape after deformation. The mechanism controlling length changes may, therefore, be composed of two elements in parallel, namely a force generating element and a passive elastic element. Inhibitors of ATP synthesis, or the presence of the non-hydrolysable ATP analogue AMP.PNP, perfused into outer hair cells, failed to inhibit length changes. Drugs against actin, including phalloidin, cytochalasin B and cytochalasin D, and against tubulin, including colchicine, nocodazole and colcemid, also failed to inhibit length changes. We conclude that the force-generating mechanism is, therefore, unlike most other forms of cell motility, and possible alternative hypotheses are briefly discussed.

Chapter 8 The Axenic Mutations and Endocytosis in Dictyostelium

The axenic mutations of Dictyostelium offer a unique opportunity to explore the relationship between phagocytosis and pinocytosis and to examine the mechanism by which a cell shifts from one mode of feeding to the other. The axenic mutations also provide a means of exploring the relationships between endocytosis and other forms of cell motility. This chapter has described the known mutations that affect axenic growth, methods for culturing wild-type and axenic cells and measuring their growth, and methods for monitoring the effects of the axenic mutations on endocytosis and cell movement. The importance has been emphasized of distinguishing effects of the axenic genotype that are expressed constitutively (i.e., during growth on either bacteria or liquid medium) from those that are a function of axenic growth conditions. The methods described in this chapter, applied to wild-type cells and to cells carrying a full complement of the axenic mutations, have shown that the axenic mutations have constitutive effects on cell-substratum interactions, and inducible effects on cell locomotion and pinocytosis.

Identification of kinesin in sea urchin eggs, and evidence for its localization in the mitotic spindle.

To understand the molecular basis of microtubule-associated motility during mitosis, the mechanochemical factors that generate the relevant motile force must be identified. Myosin, the ATPase that interacts with actin to produce the force for muscle contraction and other forms of cell motility, is believed to be involved in cytokinesis but not in mitosis. Dynein, the mechanochemical enzyme that drives microtubule sliding in eukaryotic cilia and flagella, has been identified in the cytoplasm of sea urchin eggs, but the evidence that it is involved in cytoplasmic microtubule-based motility (rather than serving as a precursor for embryonic cilia) is equivocal. Microtubule-associated ATPases have been prepared from other tissues, but their role in cytoplasmic motility is also unknown. Recent work on axoplasmic transport, however, has led to the identification of a novel mechanochemical protein called kinesin, which is thought to generate the force for moving vesicles along axonal microtubules. These results suggest that kinesin may also be a mechanochemical factor for non-axoplasmic forms of microtubule-based motility, such as mitosis. We describe here the identification and isolation of a kinesin-like protein from the cytoplasm of sea urchin eggs. We present evidence that this protein is localized in the mitotic spindle, and propose that it may be a mechanochemical factor for some form of motility associated with the mitotic spindle.

Movement of myosin-coated beads on oriented filaments reconstituted from purified actin

Although the biochemical properties of the actin/myosin interaction have been studied extensively using actin activation of myosin ATPase as an assay, until recently no well-defined assay has been available to measure the mechanical properties of ATP-dependent movement of myosin along actin filaments. The first direct measurements of the rate of myosin movement in vitro used a naturally occurring, biochemically ill-defined array of actin filaments from the alga Nitella. We report here the construction of an oriented array of filaments reconstituted from purified muscle actin and the use of this array in a biochemically defined quantitative assay for the directed movement of myosin-coated polystyrene beads. We demonstrate for the first time that actin alone, linked to a substratum by a protein anchor, is sufficient to support movement of myosin at rates consistent with the speeds of muscle contraction and other forms of cell motility.

Potent inhibition of dynein adenosinetriphosphatase and of the motility of cilia and sperm flagella by vanadate.

The motility of demembranated sea urchin sperm flagella and that of embryo cilia reactivated with 0.1 mM ATP are completely inhibited by 4 micron and 0.5 micron vanadium(V) [V(V), in vanadate], respectively. The Mg2+-activated ATPase activity (ATP phosphohydrolase, EC 3.6.1.3)of the latent form of dynein 1 is inhibited 50% by 0.5-1 micron V(V), while the Ca2+-activated ATPase activity is much less sensitive. The inhibition of flagellar beat frequency and of dynein 1 ATPase activity by V(V) appears not to be competitive with ATP. In agreement with other reports, the inhibition of (Na,K)-ATPase by V(V) shows a slow onset in the presence of ATP and is relatively rapid in its absence. With dynein, however, the inhibition occurs at a rapid rate whether or not ATP is present. Catechol at a concentration of 1 mM reverses the V(V) inhibition of reactivated sperm motility, dynein ATPase, and (Na, K)-ATPase. Myosin and actomyosin ATPases show no inhibition by concentrations of V(V) up to 500 micron. The inhibition by V(V) provides a possible technique for distinguishing between the actions of dynein and myosin in different forms of cell motility.

On the molecular basis of action of cytochalasin B.

AbstractTritium‐labeled cytochalasin B binds rapidly and reversibly to mammalian cells, and a class of high‐affinity sites (Kn ≅ 10−7 M) and a class of low‐affinity sites (KD ⩾ 10−5 M) are detected. In red blood cells, the high‐affinity binding sites (about 3 × 105 per cell) are associated with the plasma membrane, and at least 80% of these appear to be intimately related to the glucose transport system. Fractionation of cellular components of platelets by differential centrifugation and gel filtration chromatography reveals that the high‐affinity binding sites in these cells are also associated with membranous materials. A substantial number of the low‐affinity binding sites can be traced to platelet actin. The binding of cytochalasin B to actin is consistent with the alteration of intrinsic viscosity and morphology of actin filaments in vitro by the compound at concentrations of around 10−5‐10−4 M. The interaction of cytochalasin B with actin may account for its inhibitory effect on various forms of cell motility.