Relative motion inAmoeba proteus in respect to the adhesion sites

Summary The whole ectoplasmic layer of polytactic and heterotactic forms ofA. proteus behaves as self-contractile structure. Depending on the configuration of cell body and on the cell-to-substrate attachment conditions it continuously retracts from each distal cell projection toward its centre and/or from each free body end toward the actual adhesion sites. As in the monotactic forms, it leads to the withdrawal of the tail region behind the retraction center and may result in the fountain movement in front of it. In the long unattached pseudopodia of heterotactic forms the ectoplasm is retracted in the fountain form, with the velocity linearly increasing from the basis of pseudopodium up to its tip. In polytactic cells the fountain is often absent, if the advancing fronts immediately adhere to the substrate. When they develop in unattached condition, or are experimentally obliged to detach, the ectoplasmic cylinders of frontal pseudopodia are retracted backwards. On the substrates which do not offer firm points of support the cell periphery moves back as a whole,i.e., the principal ectoplasmic cylinder retracts together with the cylinders of lateral pseudopodia, and the direction and speed of movement in any spot is the resultant of forces produced by all other segments. The retraction of ectoplasmic gel layer is independent of the endoplasmic flow in such extent that a pseudopodium may be withdrawn as a whole in spite of the endoplasm streaming directed forwards in its interior. On the cell surface the particles attached by adhesion (glass rods) strictly follow the movements of the internal ectoplasmic structures, whereas the unattached particles flow forward in the direction of endoplasm streaming.Study supported by Research Project II. 1 of the Polish Academy of Science.     

Velocity distribution of the anterograde and retrograde transport of extracellular particles byAmoeba proteus

Summary The transverse velocity profiles of the anterograde flow of particles on the cell surface and around it are approximately parabolic. The peak velocity is recorded close to the membrane and the descendent arm of the profile is viscosity-dependent. It indicates that the extracellular forward flow is probably generated by a forward movement of the fluid fraction of the membrane itself. The retrograde component of extracellular movements is manifested by particles kept on the cell surface by adhesion, which behave exactly as the ectoplasmic layer on the opposite side of the membrane,i.e., they probably reflect the movement of that fraction of the surface material which is attached to the cortical microfilaments. In the longitudinal profile, the velocity of anterograde flow rises from the tail to the front of amoeba, but is generally related to the effective cell locomotion rate and not to the movements of any intracellular layer. Around the cells deprived of any attachment to the substratum, which cannot locomote but manifest vigorous intracellular movements, the anterograde flow ceases at least along 2/3 of their lenght. It persists, however, around the frontal fountain zone, where other particles still move backwards together with the retracted ectoplasmic layer. This indicates that the role of the forward flow of and on the cell surface is to compensate for: (1) the increase of the surface area in the frontal regions due to locomotion, (2) the withdrawal of a part of material which is hauled back by the retracting cortical layer. A comprehensive scheme of the velocity distribution within the different layers of a moving amoeba and around it has been constructed on the basis of present and earlier data.Study supported by the Research Project CPBP 04.01 of the Polish Academy of Science.I dedicate this paper to Professor K. E. Wohlfarth-Bottermann with the best wishes for his 65th birthday.     

Electron Microscopy of the Ectoplasm and the Proboscis in Didinium nasutum

SYNOPSIS. A morphological study on the ectoplasm and the proboscis in the ciliate Didinium nasutum , has been performed by means of an electron microscope. The ectoplasm and the endoplasm of Didinium are separated by a fibrous layer. In addition to the ciliary apparatus and the filament system, the ectoplasm is characterized by having ectoplasmic vacuoles enclosing cross-striated bodies and by having small rods surrounding the ciliary basal body.
The filament system is composed of 4 types of tubular filaments: primary filaments originating from the basal body, secondary ones coursing longitudinally along the cell periphery, tertiary ones going down in cylindrical arrays from the periphery of the proboscis into the endoplasm, and finally kinetosomal ones from the base of the basal body into the endoplasm through the newly found pore of the fibrous layer.
The fine morphology of the trichites in the proboscis is elucidated three-dimensionally and illustrated schematically. Moreover, the correlation among the small rod, ectoplasmic vacuole and trichite is discussed.     

Rapid retraction of neurites by sensory neurons in response to increased concentrations of nerve growth factor

The phenomenon of growth cone (GC) and neurite retraction resulting from a rapid incrase in concentration of the trophic molecule NGF was studied. Neurite outgrowth from explants of 8-d chick embryo dorsal root ganglia was achieved at very low NGF concentrations with heart conditioned medium during overnight culture. Quickly incrasing the NGF concentration in the growth medium dramatically affected GC and neurite morphology: the majority of GCs and neurites collapsed and retracted towards the cell body over a course of approximately 2-5 min. Retraction was elicited by increasing NGF levels from 0 to 0.05 ng/ml to as little as 0.5 ng/ml but did not occur if the NGF concentration during the initial overnight culture period exceeded 0.8 ng/ml, regardless of how much the concentration was elevated. Similar concentration changes of cytochrome c or insulin did nt result in retraction. Neurites that had been separated from their cell bodies by cutting close to their exit from the explant still retracted when NGF levels were raised. Cytochalasin B reversible inhibits retraction, whereas colchicine allows retraction to occur. Observation of cell- substratum adhesion during retraction revealed that some adhesion points remain during retraction and that they correspond to the ends of NGF leels and that it may involve microfilaments in the neurite cytoskeleton. The NGF concentration changes that elicit neurite retraction suggest that a primary event in retraction may be increased occupancy of a high-affinity NGF receptor on neurites.     

Reorganization and translocation of the ectoplasmic cytoskeleton in the leech zygote by condensation of cytasters and interactions of dynamic microtubules and actin filaments

The formation and bipolar translocation of an ectoplasmic cytoskeleton of rings and meridional bands was studied in interphase zygotes of the glossiphoniid leech Theromyzon trizonare. Zygotes consisted of a peripheral organelle-rich ectoplasm and an internal yolk-rich endoplasm. After microinjection of labeled tubulin and/or actin, zygotes were examined by time-lapse video imaging, immunofluorescence and confocal microscopy. The rings and meridional bands were formed by condensation of a network of moving cytasters that represented ectoplasmic secondary centers of microtubule and actin filament nucleation. In some cases the network of cytasters persisted between the rings. The cytoskeleton had an outer actin layer and an inner microtubule layer that merged at the irregularly-shaped boundary zone. Bipolar translocation of the rings, meridional bands, or the network of cytasters led to accumulation of the cytoskeleton at both zygote poles. Translocation of the cytoskeleton was slowed or arrested by microinjected taxol or phalloidin, in a dose-dependent fashion. Results of drug treatment probably indicate differences in the degree and speed at which the cytoskeleton becomes stabilized. Moreover, drugs that selectively stabilized either microtubules or actin filaments stabilized and impaired movement of the entire cytoskeleton. Microtubule poisons and latrunculin-B failed to disrupt the cytoskeleton. It is concluded that the microtubule and actin cytoskeletons are dynamic, presumably cross-linked and resistant to depolymerizing drugs. They probably move along each other by a sliding mechanism that depends on the instability of microtubules and actin filaments.     

Dynamics of the actin-binding protein drebrin in motile cells and definition of a juxtanuclear drebrin-enriched zone

The actin-binding protein (ABP) drebrin, isoform E2, is involved in remodelling of the actin cytoskeleton and in formation of cell processes, but its role in cell migration has not yet been investigated. Therefore, we have studied the organization of drebrin in motile cultured cells such as murine B16F1 melanoma and human SV80 fibroblast cells, using live cell confocal microscopy. In cells overexpressing DNA constructs encoding drebrin linked to EGFP, numerous long, branched cell processes were formed which slowly retracted and extended, whereas forward movement was halted. In contrast, stably transfected B16F1 cells containing drebrin-EGFP at physiological levels displayed lamellipodia and were able to migrate on laminin. Surprisingly, in such cells, drebrin was absent from anterior lamellipodia but was enriched in a specific juxtanuclear zone, the "drebrin-enriched zone" (DZ), and in the tail. In leading edges of SV80 cells, characterized by pronounced actin microspikes, drebrin was specifically enriched along posterior portions of the microspikes, together with tropomyosin. Drebrin knock-down by small interfering RNAs did not impair movements of SV80 cells. Our results confirm the role of drebrin E2 in the formation of branching processes and further indicate that during cell migration, the protein contributes to retraction of the cell body and the tail but not to lamellipodia formation. In particular, the novel, sizable juxtanuclear DZ structure will have to be characterized in future experiments with respect to its molecular assembly and cell biological functions.     

The contractile basis of amoeboid movement : IV. The viscoelasticity and contractility of amoeba cytoplasm in vivo

The viscoelasticity and contractility of amoeba cytoplasm has been studied in vivo and in vitro. A gradient of increasing viscoelasticity and contractility was identified in the endoplasm of intact cells from the uroid (tail) to the fountain zone (tip of advancing pseudopod). Anterior endoplasm, as well as all of the ectoplasm, contracted in response to the microinjection of a threshold calcium ion concentration (ca 7.0 × 10⁻⁷ M). In contrast, there were only delayed weak contractions in the uroid endoplasm upon the microinjection of a threshold calcium ion concentration. Contractions induced in the ectoplasm by microinjecting the contraction solution readily caused the endoplasm to stream. However, the endoplasm at the tips of the extending pseudopods were also contractile and transmitted applied tensions. Furthermore, the microinjection of subthreshold calcium ion concentrations caused the loss of distinct endoplasmic structure and the cessation of streaming in both the uroid and the anterior third of the cell. In addition, the relationship between contractility and cytoplasmic streaming was characterized in “relaxed” cytoplasm placed in a gradient of calcium ion concentration inside quartz capillaries. The results of these experiments demonstrated that the mechanochemical conversion of endoplasm to ectoplasm caused the cytoplasm to become more structured and contractile. Therefore, physiological contractions are possible during and after the conversion of endoplasm to ectoplasm.     

Mechanics and control of the cytoskeleton in Amoeba proteus.

Many models of the cytoskeletal motility of Amoeba proteus can be formulated in terms of the theory of reactive interpenetrating flow (Dembo and Harlow, 1986). We have devised numerical methodology for testing such models against the phenomenon of steady axisymmetric fountain flow. The simplest workable scheme revealed by such tests (the minimal model) is the main preoccupation of this study. All parameters of the minimal model are determined from available data. Using these parameters the model quantitatively accounts for the self assembly of the cytoskeleton of A. proteus: for the formation and detailed morphology of the endoplasmic channel, the ectoplasmic tube, the uropod, the plasma gel sheet, and the hyaline cap. The model accounts for the kinematics of the cytoskeleton: the detailed velocity field of the forward flow of the endoplasm, the contraction of the ectoplasmic tube, and the inversion of the flow in the fountain zone. The model also gives a satisfactory account of measurements of pressure gradients, measurements of heat dissipation, and measurements of the output of useful work by amoeba. Finally, the model suggests a very promising (but still hypothetical) continuum formulation of the free boundary problem of amoeboid motion. by balancing normal forces on the plasma membrane as closely as possible, the minimal model is able to predict the turgor pressure and surface tension of A. proteus. Several dynamical factors are crucial to the success of the minimal model and are likely to be general features of cytoskeletal mechanics and control in amoeboid cells. These are: a constitutive law for the viscosity of the contractile network that includes an automatic process of gelation as the network density gets large; a very vigorous cycle of network polymerization and depolymerization (in the case of A. proteus, the time constant for this reaction is approximately 12 s); control of network contractility by a diffusible factor (probably calcium ion); and control of the adhesive interaction between the cytoskeleton and the inner surface of the plasma membrane.     

Effect of osmolality and selected ions on retraction of the distome body into the cercaria tail chamber of Proterometra macrostoma (Trematoda: Azygiidae)

The furcocystocercous cercariae of the digenetic trematode, Proterometra macrostoma , possess a tail chamber into which their distome body withdraws prior to emergence from their snail intermediate host. The process of distome retraction and the conditions that trigger it in this species are not clear. The objectives of the present study were (1) to describe the retraction process in P. macrostoma; (2) to assess whether osmolality affects cercarial retraction; (3) to evaluate the effect of selected ions on retraction; and (4) to compare the swimming effectiveness of naturally (?= in vivo) retracted versus in vitro retracted cercariae. Retraction of the cercaria body into its tail chamber required only 2 min or less once initiated. The process began with the development of a chamber within the anterior end of the worm's tail. The chamber's lip advanced in a pulsating motion over the stationary distome. Retraction was completed with the constriction and fusion of the chamber lip once it passed over the anterior end of the distome, sealing the latter within the tail chamber. There was a significant difference in the proportions of cercariae with bodies retracted into tails, bodies not retracted, and bodies separated from tails in artificial pond water (APW) versus artificial snail water (ASW). A greater number of cercariae withdrew into their tail chambers in ASW (59/124; 47.6%) than in APW (21/124; 16.9%). In APW, more bodies separated from their tails (24/124; 19.4%) than in ASW (3/124; 2.4%). In both solutions (APW: 63.7% = 79/124; ASW: 50% = 62/124), a majority of cercariae never retracted. In APW, 76.2% of distomes retracting into their tails did so within the first 5 min compared to only 30.5% in ASW. There was no significant difference in the proportions of cercariae with bodies retracted into tails, bodies not retracted, and bodies separated from tails based on isosmotic replacement of individual ions, i.e., Na(+), K(+), Ca(++), or Mg(++), in ASW with Li(+). There was also no significant difference in the vertical swimming burst distance in cercariae whose bodies were initially retracted into their tails in vitro versus in vivo.     

Localized depolymerization of the major sperm protein cytoskeleton correlates with the forward movement of the cell body in the amoeboid movement of nematode sperm.

The major sperm protein (MSP)-based amoeboid motility of Ascaris suum sperm requires coordinated lamellipodial protrusion and cell body retraction. In these cells, protrusion and retraction are tightly coupled to the assembly and disassembly of the cytoskeleton at opposite ends of the lamellipodium. Although polymerization along the leading edge appears to drive protrusion, the behavior of sperm tethered to the substrate showed that an additional force is required to pull the cell body forward. To examine the mechanism of cell body movement, we used pH to uncouple cytoskeletal polymerization and depolymerization. In sperm treated with pH 6.75 buffer, protrusion of the leading edge slowed dramatically while both cytoskeletal disassembly at the base of the lamellipodium and cell body retraction continued. At pH 6.35, the cytoskeleton pulled away from the leading edge and receded through the lamellipodium as its disassembly at the cell body continued. The cytoskeleton disassembled rapidly and completely in cells treated at pH 5.5, but reformed when the cells were washed with physiological buffer. Cytoskeletal reassembly occurred at the lamellipodial margin and caused membrane protrusion, but the cell body did not move until the cytoskeleton was rebuilt and depolymerization resumed. These results indicate that cell body retraction is mediated by tension in the cytoskeleton, correlated with MSP depolymerization at the base of the lamellipodium.     

Immunodetection and intracellular localization of caldesmon-like proteins in Amoeba proteus

Summary. Caldesmon immunoanalogues were detected in Amoeba proteus cell homogenates by the Western blot technique. Three immunoreactive bands were recognized by polyclonal antibodies against the whole molecule of chicken gizzard caldesmon as well as by a monoclonal antibody against its C-terminal domain: one major and two minor bands corresponding to proteins with apparent molecular masses of 150, 69, and 60 kDa. The presence of caldesmon-like protein(s) in amoebae was revealed as well in single cells after their fixation, staining with the same antibodies, and recording their total fluorescence in a confocal laser scanning microscope. Proteins recognized by the antibodies bind to filamentous actin. This was established by a cosedimentation assay in cell homogenates and by colocalization of the caldesmon-related immunofluorescence with the fluorescence of filamentous actin stained with rhodamine-labelled phalloidin, demonstrated in optical sections of single cells in a confocal microscope. Caldesmon is colocalized with filamentous actin in the withdrawn cell regions where the cortical actomyosin network contracts and actin is depolymerized, in the frontal zone where actin is polymerized again and the cortical cytoskeleton is reconstructed, inside the nucleus and in the perinuclear cytoskeleton, and probably at the cell-to-substratum adhesion sites. The regulatory role of caldesmon in these functionally different regions of locomoting amoebae is discussed.Correspondence and reprints: Department of Cell Biology, Nencki Institute of Experimental Biology, ulica Pasteura 3, 02-093 Warsaw, Poland.Received October 7, 2002; accepted December 2, 2002; published online August 26, 2003     

Integrating the actin and vimentin cytoskeletons. adhesion-dependent formation of fimbrin-vimentin complexes in macrophages.

Cells adhere to the substratum through specialized structures that are linked to the actin cytoskeleton. Recent studies report that adhesion also involves the intermediate filament (IF) and microtubule cytoskeletons, although their mechanisms of interaction are unknown. Here we report evidence for a novel adhesion-dependent interaction between components of the actin and IF cytoskeletons. In biochemical fractionation experiments, fimbrin and vimentin coprecipitate from detergent extracts of macrophages using vimentin- or fimbrin-specific antisera. Fluorescence microscopy confirms the biochemical association. Both proteins colocalized to podosomes in the earliest stages of cell adhesion and spreading. The complex is also found in filopodia and retraction fibers. After detergent extraction, fimbrin and vimentin staining of podosomes, filopodia, and retraction fibers are lost, confirming that the complex is localized to these structures. A 1:4 stoichiometry of fimbrin binding to vimentin and a low percentage (1%) of the extracted vimentin suggest that fimbrin interacts with a vimentin subunit. A fimbrin-binding site was identified in the NH(2)-terminal domain of vimentin and the vimentin binding site at residues 143-188 in the CH1 domain of fimbrin. Based on these observations, we propose that a fimbrin-vimentin complex may be involved in directing the assembly of the vimentin cytoskeleton at cell adhesion sites.     

Human liver sinusoidal endothelial cells respond to interaction with Entamoeba histolytica by changes in morphology, integrin signalling and cell death

Invasive infection with Entamoeba histolytica causes intestinal and hepatic amoebiasis. In liver, parasites cross the endothelial barrier before abscess formation in the parenchyma. We focussed on amoebae interactions with human hepatic endothelial cells, the latter potentially playing a dual role in the infection process: as a barrier and as modulators of host defence responses. We characterized early responses of a human liver sinusoidal endothelial cell line to virulent and virulence-attenuated E. histolytica. Within the first minutes human cells start to retract, enter into apoptosis and die. In the presence of virulent amoebae, expression of genes related to cell cycle, cell death and integrin-mediated adhesion signalling was modulated, and actin fibre, focal adhesion kinase and paxillin localizations changed. Effects of inhibitors and amoeba strains not expressing pathogenic factors amoebapore A and cysteine protease A5 indicated that cell death and cytoskeleton disorganization depend upon parasite adhesion and amoebic cysteine proteinase activities. The data establish a relation between cytotoxic effects of E. histolytica and altered human target cell adhesion and suggest that interference with adhesion signalling triggers endothelial cell retraction and death. Understanding the roles of integrin signalling in endothelial cells will provide clues to unravel host-pathogen interactions during amoebic liver infection.     

Actin,microtubules and focal adhesion dynamics during cell migration

Cell migration is a complex cellular behavior that results from the coordinated changes in the actin cytoskeleton and the controlled formation and dispersal of cell-substrate adhesion sites. While the actin cytoskeleton provides the driving force at the cell front, the microtubule network assumes a regulatory function in coordinating rear retraction. The polarity within migrating cells is further highlighted by the stationary behavior of focal adhesions in the front and their sliding in trailing ends. We discuss here the cross-talk of the actin cytoskeleton with the microtubule network and the potential mechanisms that control the differential behavior of focal adhesions sites during cell migration.     

The C terminus of talin links integrins to cell cycle progression

Integrins are cell adhesion receptors that sense the extracellular matrix (ECM) environment. One of their functions is to regulate cell fate decisions, although the question of how integrins initiate intracellular signaling is not fully resolved. In this paper, we examine the role of talin, an adapter protein at cell-matrix attachment sites, in outside-in signaling. We used lentiviral small hairpin ribonucleic acid to deplete talin in mammary epithelial cells. These cells still attached to the ECM in an integrin-dependent manner and spread. They had a normal actin cytoskeleton, but vinculin, paxillin, focal adhesion kinase (FAK), and integrin-linked kinase were not recruited to adhesion sites. Talin-deficient cells showed proliferation defects, and reexpressing a tail portion of the talin rod, but not its head domain, restored integrin-mediated FAK phosphorylation, suppressed p21 expression, and rescued cell cycle. Thus, talin recruits and activates focal adhesion proteins required for proliferation via the C terminus of its rod domain. Our study reveals a new function for talin, which is to link integrin adhesions with cell cycle progression.     

In vitro models of tail contraction and cytoplasmic streaming in amoeboid cells

We have developed a reconstituted gel-sol and contractile model system that mimics the structure and dynamics found at the ectoplasm/endoplasm interface in the tails of many amoeboid cells. We tested the role of gel-sol transformations of the actin-based cytoskeleton in the regulation of contraction and in the generation of endoplasm from ectoplasm. In a model system with fully phosphorylated myosin II, we demonstrated that either decreasing the actin filament length distribution or decreasing the extent of actin filament cross-linking initiated both a weakening of the gel strength and contraction. However, streaming of the solated gel components occurred only under conditions where the length distribution of actin was decreased, causing a self-destruct process of continued solation and contraction of the gel. These results offer significant support that gel strength plays an important role in the regulation of actin/myosin II-based contractions of the tail cortex in many amoeboid cells as defined by the solation-contraction coupling hypothesis (Taylor, D. L., and M. Fechheimer. 1982. Phil. Trans. Soc. Lond. B. 299:185-197). The competing processes of solation and contraction of the gel would appear to be mutually exclusive. However, it is the temporal-spatial balance of the rate and extent of two stages of solation, coupled to contraction, that can explain the conversion of gelled ectoplasm in the tail to a solated endoplasm within the same small volume, generation of a force for the retraction of tails, maintenance of cell polarity, and creation of a positive hydrostatic pressure to push against the newly formed endoplasm. The mechanism of solation-contraction of cortical cytoplasm may be a general component of the normal movement of a variety of amoeboid cells and may also be a component of other contractile events such as cytokinesis.     

Motion of the Longitudinal Flagellum in Ceratium tripos (Dinoflagellida): A Retractile Flagellar Motion1

The longitudinal flagellum of Ceratium tripos moves in two dissimilar ways: undulation and retraction. The undulatory wave is planar and has a wavelength of 74.3 ± 9.6 μm and an amplitude of 14.2 ± 2.3 μm in sea water. The beat frequency is 30 Hz at 20°C, pH 8.0. The retractile motion is unique to Ceratium and is triggered by mechanical stimulation on the cell body, especially at the tip of the apical horn. When it retracts, the longitudinal flagellum folds every 4–5 μm along the flagellum. Cinematographic study showed that the flagellum folded from tip to base and was finally installed into the sulcus, a groove on the ventral side of the cell. This motion is completed in sea water within 28 msec. The retracted flagellum then re-extends and restores the undulation within a few seconds. The flagellum unfolds in the proximal portion first, then the distal, and finally the middle portion. Fixation always triggers the retraction. Scanning electron microscopy showed that the flagellum is folded and secondarily twisted in a helix. A new fiber in addition to the flagellar axoneme was found in the retracted flagellum by phase microscopy. This fiber (R-fiber) seems to contract during the retraction to fold the flagellum.     

Patterning of the membrane cytoskeleton by the extracellular matrix

The extracellular matrices of different tissues contain components which affect the migration, morphology and differentiation of many types of cells. These forms of cell behavior often involve dramatic changes in cytoskeletal organization. Extracellular matrix components are recognized by specific cell surface receptors which span the membrane and interact with the actin cytoskeleton. In cultured cells, the matrix receptors are concentrated in sites of cell attachment called focal adhesions. Information that is conveyed from the extracellular matrix to the cytoskeleton may involve matrix components, cell surface receptors, as well as the proteins at the cytoplasmic face of the focal adhesion which link the receptors to the actin cytoskeleton.     

Traction force microscopy in rapidly moving cells reveals separate roles for ROCK and MLCK in the mechanics of retraction

Retraction is a major rate-limiting step in cell motility, particularly in slow moving cell types that form large stable adhesions. Myosin II dependent contractile forces are thought to facilitate detachment by physically pulling up the rear edge. However, retraction can occur in the absence of myosin II activity in cell types that form small labile adhesions. To investigate the role of contractile force generation in retraction, we performed traction force microscopy during the movement of fish epithelial keratocytes. By correlating changes in local traction stress at the rear with the area retracted, we identified four distinct modes of retraction. “Recoil” retractions are preceded by a rise in local traction stress, while rear edge is temporarily stuck, followed by a sharp drop in traction stress upon detachment. This retraction type was most common in cells generating high average traction stress. In “pull” type retractions local traction stress and area retracted increase concomitantly. This was the predominant type of retraction in keratocytes and was observed mostly in cells generating low average traction stress. “Continuous” type retractions occur without any detectable change in traction stress, and are seen in cells generating low average traction stress. In contrast, to many other cell types, “release” type retractions occur in keratocytes following a decrease in local traction stress. Our identification of distinct modes of retraction suggests that contractile forces may play different roles in detachment that are related to rear adhesion strength. To determine how the regulation of contractility via MLCK or Rho kinase contributes to the mechanics of detachment, inhibitors were used to block or augment these pathways. Modulation of MLCK activity led to the most rapid change in local traction stress suggesting its importance in regulating attachment strength. Surprisingly, Rho kinase was not required for detachment, but was essential for localizing retraction to the rear. We suggest that in keratocytes MLCK and Rho kinase play distinct, complementary roles in the respective temporal and spatial control of rear detachment that is essential for maintaining rapid motility.