Protrusion Velocity Research Articles

Nascent adhesions shorten the period of lamellipodium protrusion through the Brownian ratchet mechanism.

Directional cell migration is driven by the conversion of oscillating edge motion into lasting periods of leading edge protrusion. Actin polymerization against the membrane and adhesions control edge motion, but the exact mechanisms that determine protrusion period remain elusive. We addressed this by developing a computational model in which polymerization of actin filaments against a deformable membrane and variable adhesion dynamics support edge motion. Consistent with previous reports, our model showed that actin polymerization and adhesion lifetime power protrusion velocity. However, increasing adhesion lifetime decreased the protrusion period. Measurements of adhesion lifetime and edge motion in migrating cells confirmed that adhesion lifetime is associated with and promotes protrusion velocity, but decreased duration. Our model showed that adhesions' control of protrusion persistence originates from the Brownian ratchet mechanism for actin filament polymerization. With longer adhesion lifetime or increased-adhesion density, the proportion of actin filaments tethered to the substrate increased, maintaining filaments against the cell membrane. The reduced filament-membrane distance generated pushing force for high edge velocity, but limited further polymerization needed for protrusion duration. We propose a mechanism for cell edge protrusion in which adhesion strength regulates actin filament polymerization to control the periods of leading edge protrusion.

Re-track: Software to analyze the retraction and protrusion velocities of neurites, filopodia and other structures

We have developed new software, Re-track, that will quantify the rates of retraction and protrusion of structures emanating from the central core of a cell, such as neurites or filopodia. Re-Track, uses time-lapse images of cells in TIFF format and calculates the velocity of retraction or protrusion of a selected structure. The software uses a flexible moving boundary and has the ability to correct this boundary throughout analysis. Re-Track is fast, platform independent, and user friendly, and it can be used to follow biological events such as changes in neuronal connections, tip-growing cells such as moss, adaptive migration of cells, and similar behavior in non-biological systems.

Monitoring the Maturation of Crambe Seeds Using X-ray Image Analysis

The application of modern and efficient techniques to access seed physical and physiological information has contributed to new advances in the agricultural sector. The objective of this study was to evaluate morphometric parameters obtained from the X-Ray analysis using crambe seeds harvested at different maturation stages and to relate them to seed physiological potential. Seeds harvested at different maturation stages were evaluated using X-Ray test, germination, first germination count, germination speed index, root protrusion velocity and accelerated aging. The Principal Components Analysis was applied to data collected. The results demonstrated that the X-Ray analysis allowed to visualize internal morphology of crambe seeds. Differences between seeds harvested at different maturation levels were observed. Correlations between physical variables were verified, such as seed area with germination (r = 0.96). The use of X-Ray analysis combined with semi-automated analysis is efficient to differentiate crambe seeds in different maturation stages. The morphometric parameters are related to the physiological quality of crambe seeds.

On the relation between filament density, force generation, and protrusion rate in mesenchymal cell motility.

Lamellipodia are flat membrane protrusions formed during mesenchymal motion. Polymerization at the leading edge assembles the actin filament network and generates protrusion force. How this force is supported by the network and how the assembly rate is shared between protrusion and network retrograde flow determines the protrusion rate. We use mathematical modeling to understand experiments changing the F-actin density in lamellipodia of B16-F1 melanoma cells by modulation of Arp2/3 complex activity or knockout of the formins FMNL2 and FMNL3. Cells respond to a reduction of density with a decrease of protrusion velocity, an increase in the ratio of force to filament number, but constant network assembly rate. The relation between protrusion force and tension gradient in the F-actin network and the density dependency of friction, elasticity, and viscosity of the network explain the experimental observations. The formins act as filament nucleators and elongators with differential rates. Modulation of their activity suggests an effect on network assembly rate. Contrary to these expectations, the effect of changes in elongator composition is much weaker than the consequences of the density change. We conclude that the force acting on the leading edge membrane is the force required to drive F-actin network retrograde flow.

FMNL formins boost lamellipodial force generation

Migration frequently involves Rac-mediated protrusion of lamellipodia, formed by Arp2/3 complex-dependent branching thought to be crucial for force generation and stability of these networks. The formins FMNL2 and FMNL3 are Cdc42 effectors targeting to the lamellipodium tip and shown here to nucleate and elongate actin filaments with complementary activities in vitro. In migrating B16-F1 melanoma cells, both formins contribute to the velocity of lamellipodium protrusion. Loss of FMNL2/3 function in melanoma cells and fibroblasts reduces lamellipodial width, actin filament density and -bundling, without changing patterns of Arp2/3 complex incorporation. Strikingly, in melanoma cells, FMNL2/3 gene inactivation almost completely abolishes protrusion forces exerted by lamellipodia and modifies their ultrastructural organization. Consistently, CRISPR/Cas-mediated depletion of FMNL2/3 in fibroblasts reduces both migration and capability of cells to move against viscous media. Together, we conclude that force generation in lamellipodia strongly depends on FMNL formin activity, operating in addition to Arp2/3 complex-dependent filament branching.

Measurement of Lamellipodial Protrusion by Optical Trapping

Lamellipodial protrusion is a fundamental step in cell locomotion. This important process is driven by actin polymerization, but its physical aspect has not yet been fully investigated. We previously studied the lamellipodial protrusion of Swiss 3T3 fibroblast cells at 33 ms temporal resolution and 10 nm accuracy by probing the motion of the cell edge with a 1 µm bead held in an optical trap. In that study, we found a transient mode of protrusion and analyzed the time variation of the protrusion velocity. In this study, we analyzed the power spectra of the fluctuations of a trap-held bead during the protrusion and found cell-specific fluctuations. The maximal amplitude of the fluctuations was 23 nm, which was estimated from the power of the fluctuations accumulated over 1.9 and 7.6 Hz. This value was significantly larger than that of the fluctuations of the trap-held bead that is not in contact with the cell edge (14 nm). The amplitude of the fluctuation of the probing bead showed a positive correlation w...

Morphology and fibre-type distribution in the tongue of the Pogona vitticeps lizard (Iguania, Agamidae).

Agamid lizards use tongue prehension for capturing all types of prey. The purpose of this study was to investigate the functional relationship between tongue structure, both surface and musculature, and function during prey capture in Pogona vitticeps. The lack of a detailed description of the distribution of fibre-types in the tongue muscles in some iguanian lizards has hindered the understanding of the functional morphology of the lizard tongue. Three methodological approaches were used to fill this gap. First, morphological analyses were performed (i) on the tongue surface through scanning electron microscopy, and (ii) on the lingual muscle by histological coloration and histochemistry to identify fibre-typing. Secondly, kinematics of prey capture was quantified by using high-speed video recordings to determine the movement capabilities of the tongue. Finally, electromyography (EMG) was used to identify the motor pattern tongue muscles during prey capture. Morphological and functional data were combined to discuss the functional morphology of the tongue in agamid lizards, in relation to their diet. During tongue protraction, M. genioglossus contracts 420 ± 96 ms before tongue-prey contact. Subsequently, Mm. verticalis and hyoglossus contract throughout tongue protraction and retraction. Significant differences are found between the timing of activity of the protractor muscles between omnivorous agamids (Pogona sp., this study) and insectivorous species (Agama sp.), despite similar tongue and jaw kinematics. The data confirm that specialisation toward a diet which includes more vegetal materials is associated with significant changes in tongue morphology and function. Histoenzymology demonstrates that protractor and retractor muscles differ in fibre composition. The proportion of fast glycolytic fibres is significantly higher in the M. hyoglossus (retractor muscle) than in the M. genioglossus (protractor muscle), and this difference is proposed to be associated with differences in the velocity of tongue protrusion and retraction (5 ± 5 and 40 ± 13 cm s(-1) , respectively), similar to Chamaeleonidae. This study provides a way to compare fibre-types and composition in all iguanian and scleroglossan lizards that use tongue prehension to catch prey.

The Role of Arp2/3 in DRG Growth Cones Motility

The Arp2/3 complex simultaneously controls nucleation of actin polymerization and filament branching. We used optical tweezers and video imaging to analyse the role of Arp2/3 in the motility of lamellipodia from developing growth cones (GCs) of isolated Dorsal Root Ganglia (DRG) neurons. Kymograph analysis shows that inhibition of Arp 2/3 complex with 100 µM CK548 completely abolished the lamellipodia motion. Surprisingly when the activity of Arp2/3 is inhibited by 25 µM CK548, lamellipodia shrank for 6-8 min but then reappears again and show an almost normal motility. The average period to complete protrusion and retraction cycles of lamellipodia 76 s in control, increased to 110 s after the recovery from the exposure to this drug. The drug CK548 decreased, initially, the ability of the lamellipodia to lift up in the axial direction, but after the recovery lamellipodia were able to lift 4 µm up above the coverslip as in control conditions. The force generated by lamellipodia and their maximal protrusion velocity decreased when the concentration of drug CK548 is increased from 25 to 100 µM. After the recovery of an almost normal motility, ruffles were present but had a different shape and were more fragmented and invaginated, suggesting an altered cytoskeleton. These results show that Arp 2/3 is important in maintaining the morphology and the motility of the neuronal growth cones but in neuronal lamellipodia several distinct pathways contribute to actin filament branching and to their cross-linkage.

AXIAL-SYMMETRIC MODELING AND KINEMATIC ANALYSIS OF SPREADING OF SPARSELY CULTURED FIBROBLASTS

Cell spreading plays an important role in the modulation of physiological functions such as inflammation and cancer metastasis. The Brownian ratchet model and Bell's model have been used to simulate actin dynamics and bond kinetics for focal adhesion dynamics, respectively. In the present study, these models were modified and two additional subcellular mechanisms, integrin and myosin kinetics, were incoporated. An integrin recruitment function was introduced to determine the size of a focal adhesion associated with the substrate stiffness. The relationship between myosin concentration and the actin protrusion velocity was described by a first-order differential equation. Subcellular processes, including cell protrusion, focal adhesion formation, and stress fiber formation, were integrated into an axial-symmetric biophysical model, while inputs to the model were kinematic data from time-lapse experiments. Numerical simulations of the model using the Gillespie algorithm showed that dynamics of cell spreading can be well described by the model.

Physical Model for Self-Organization of Actin Cytoskeleton and Adhesion Complexes at the Cell Front

Cell motion is driven by interplay between the actin cytoskeleton and the cell adhesions in the front part of the cell. The actin network segregates into lamellipodium and lamellum, whereas the adhesion complexes are characteristically distributed underneath the actin system. Here, we suggest a computational model for this characteristic organization of the actin-adhesion system. The model is based on the ability of the adhesion complexes to sense mechanical forces, the stick-slip character of the interaction between the adhesions and the moving actin network, and a hypothetical propensity of the actin network to disintegrate upon sufficiently strong stretching stresses. We identify numerically three possible types of system organization, all observed in living cells: two states in which the actin network exhibits segregation into lamellipodium and lamellum, whereas the cell edge either remains stationary or moves, and a state where the actin network does not undergo segregation. The model recovers the asynchronous fluctuations and outward bulging of the cell edge, and the dependence of the edge protrusion velocity on the rate of the nascent adhesion generation, the membrane tension, and the substrate rigidity.

Interplay Between Cytoskeletal Forces, Membrane Tension and Hydrostatic Pressure in Rapidly Migrating Cells

In motile cells, membrane pressure at the membrane/cytoplasm interface is balanced by the cytoskeletal forces and hydrostatic pressure. In particular, it is believed that protrusion of the lamellipodia is controlled by the balance of membrane pressure and the pressure generated by actin polymerization, while bleb formation results from the balance of membrane pressure and hydrostatic pressure. However, exact contributions of these forces to the cell shape and motion are not known. Membrane pressure is a product of tension and local curvature of the membrane; thus, to know the membrane pressure one has to know the exact three-dimensional shape of the cell. We have developed a simple approach to measure cell vertical profile and volume, which is based on the displacement of the fluorescent cell-impermeable dye dissolved in the medium. To get insight into the balance of forces at the membrane interface, we measure membrane tension (with tether assay), protrusion velocity, vertical profile, and volume dynamics in migrating fish epidermal keratocytes subjected to cytoskeletal drugs and volume perturbations. Inhibiting myosin-dependent contraction with blebbistatin resulted in irregular cell shape, and reduced protrusion velocity and membrane tension (variable and low tether forces as compared to a force distribution centered around 35-40 pN in control cells).Remarkably, hypoosmotic treatment normalized blebbistatin-treated cells by restoring their tension, protrusion velocity, and shape, and also increased tension and protrusion velocity in control cells. Vertical profile measurements showed that hypoosmotic treatment induced persistent cell swelling and substantial increase in the height of the lamellipodia.We propose that hydrostatic pressure that could be generated due to either myosin dependent contraction or osmotic gradient helps to maintains optimal vertical profile of the lamellipodium and thus cooperates with actin-dependent protrusion.Supported by SNSF, Swiss SystemsX and NCCBI.

Cooperative Dynamics of mDia1 and Arp2/3 in Basal Epithelial Cell Migration

Growing experimental evidence suggests that formin and Arp2/3 coexist with distinct molecular and functional properties in cell protrusions. However, current models of cell migration focus on Arp2/3-mediated actin assembly as the driver of lamellipodial protrusions. The roles of formins, and how formin-mediated filament assembly may be coordinated with Arp2/3-mediated assembly are unclear. Here, we investigated the roles of formin in cell migration. We found that mDia1 is present in lamellipodia of PtK1 cells. We then examined the recruitment dynamics of mDia1, Arp2/3, paxillin, and actin to the leading edge using quantitative live cell imaging. We found that mDia1, actin, and paxillin start to accumulate at maximum retraction edge velocity prior to the onset of protrusion events whereas Arp2/3 is recruited concomitantly with protrusions. After protrusions start, mDia1 and paxillin undergo transient decreases at maximum protrusion velocity while Arp2/3 and actin reach maximum with a delay of 10s. These results suggest that mDia1-mediated actin assembly initiates protrusions by promoting nascent adhesions, and fast protrusions are accompanied by transient decrease of adhesion formation and the reinforcement of actin polymerization by Arp2/3 against increasing membrane tension.

Clathrin is required for Scar/Wave-mediated lamellipodium formation

The Scar/Wave complex (SWC) generates lamellipodia through Arp2/3-dependent polymerisation of branched actin networks. In order to identify new SWC regulators, we conducted a screen in Drosophila cells combining proteomics with functional genomics. This screen identified Clathrin heavy chain (CHC) as a protein that binds to the SWC and whose depletion affects lamellipodium formation. This role of CHC in lamellipodium formation can be uncoupled from its role in membrane trafficking by several experimental approaches. Furthermore, CHC is detected in lamellipodia in the absence of the adaptor and accessory proteins of endocytosis. We found that CHC overexpression decreased membrane recruitment of the SWC, resulting in reduced velocity of protrusions and reduced cell migration. By contrast, when CHC was targeted to the membrane by fusion to a myristoylation sequence, we observed an increase in membrane recruitment of the SWC, protrusion velocity and cell migration. Together these data suggest that, in addition to its classical role in membrane trafficking, CHC brings the SWC to the plasma membrane, thereby controlling lamellipodium formation.

Clathrin is required for Scar/Wave-mediated lamellipodium formation

The Scar/Wave complex (SWC) generates lamellipodia through Arp2/3-dependent polymerisation of branched actin networks. In order to identify new SWC regulators, we conducted a screen in Drosophila cells combining proteomics with functional genomics. This screen identified Clathrin heavy chain (CHC) as a protein that binds to the SWC and whose depletion affects lamellipodium formation. This role of CHC in lamellipodium formation can be uncoupled from its role in membrane trafficking by several experimental approaches. Furthermore, CHC is detected in lamellipodia in the absence of the adaptor and accessory proteins of endocytosis. We found that CHC overexpression decreased membrane recruitment of the SWC, resulting in reduced velocity of protrusions and reduced cell migration. By contrast, when CHC was targeted to the membrane by fusion to a myristoylation sequence, we observed an increase in membrane recruitment of the SWC, protrusion velocity and cell migration. Together these data suggest that, in addition to its classical role in membrane trafficking, CHC brings the SWC to the plasma membrane, thereby controlling lamellipodium formation.

Simulation of Membrane Protrusion on Cellular Scale using Coarse-Grained Brownian Ratchet Model

Cell migration is a crucial process for many physiological and pathological events, such as embryogenesis and cancer metastasis. During cell migration, a lamellipodium is formed, which is a flat and broad membrane extension filled with a dense and highly branched actin network. Protrusion of the lamellipodial leading edge, referred to as membrane protrusion, is one of the essential cellular activities for continuous cell migration and a mechanochemical process of active membrane deformation driven by actin polymerization. To explain how membrane protrusion is driven by actin polymerization, Brownian ratchet (BR) model has been suggested on the basis of the underlying molecular mechanism. However, because the BR requires a priori load that cannot be determined without information of the cell shape. Therefore, to express the membrane protrusion on the cellular scale, we propose a new mathematical model, the coarse-grained BR (CBR), which is derived on the basis of nonequilibrium thermodynamics theory. The CBR can reproduce the BR within the limit of the quasistatic process of membrane protrusion, and can estimate the protrusion velocity consistently with an effective elastic constant that represents the state of the energy of the membrane. We perform a cellular-scale simulation of migrating keratocyte in which the proposed CBR is used for the membrane protrusion model on the the cellular scale. The results show that the protrusion velocity decreases with increasing the curvature of the leading edge and the stiffness of the substrate. These trends agreed with the other experimental results. Thus, the CBR can be considered an appropriate cellular-scale model to express the membrane protrusion and explains how mechanical factors affect membrane protrusion.

Coarse-grained Brownian ratchet model of membrane protrusion on cellular scale

Membrane protrusion is a mechanochemical process of active membrane deformation driven by actin polymerization. Previously, Brownian ratchet (BR) was modeled on the basis of the underlying molecular mechanism. However, because the BR requires a priori load that cannot be determined without information of the cell shape, it cannot be effective in studies in which resultant shapes are to be solved. Other cellular-scale models describing the protrusion have also been suggested for modeling a whole cell; however, these models were not developed on the basis of coarse-grained physics representing the underlying molecular mechanism. Therefore, to express the membrane protrusion on the cellular scale, we propose a novel mathematical model, the coarse-grained BR (CBR), which is derived on the basis of nonequilibrium thermodynamics theory. The CBR can reproduce the BR within the limit of the quasistatic process of membrane protrusion and can estimate the protrusion velocity consistently with an effective elastic constant that represents the state of the energy of the membrane. Finally, to demonstrate the applicability of the CBR, we attempt to perform a cellular-scale simulation of migrating keratocyte in which the proposed CBR is used for the membrane protrusion model on the cellular scale. The results show that the experimentally observed shapes of the leading edge are well reproduced by the simulation. In addition, The trend of dependences of the protrusion velocity on the curvature of the leading edge, the temperature, and the substrate stiffness also agreed with the other experimental results. Thus, the CBR can be considered an appropriate cellular-scale model to express the membrane protrusion on the basis of its underlying molecular mechanism.

The Effect of the Interaction Between the Myosin-X Ferm Domain and Integrin on Filopodia Protrusion

Filopodia are thin actin-rich plasma membrane structures found at the leading edge of migrating cells. Filopodia protrusions are regulated in part by myosin X, an unconventional myosin with a FERM domain that interacts with the adhesion factor integrin-β.We have previously found the long filopodia needed for cell adhesion were produced by having filopodia repeat cycles of short (∼3um) extensions and retractions (phased elongation). This phased elongation could be suppressed by deleting the FERM domain. This result indicates that filopodia protrusion mediated by myosin-X is strongly responsible for cell adhesion.To further investigate the importance of the FERM domain, we have examined the effects of substrate coating on filopodia. Two types of substrate coating, fibronectin and poly-lysine, were used. Fibronectin is an extracellular matrix glycoprotein that binds to integrins (integrin dependent), while poly-lysine is a synthetic molecules used to enhance cell attachment to plastic and glass surfaces (integrin independent). We found that the protrusion velocity of filopodia on fibronectin was less than that on poly-lysine, which is likely due to friction between the FERM domain and integrin.Also, the filopodia length of one elongation (one extension and retraction) on fibronectin was 1.5 fold longer than that on poly-lysine. However, this was compensated for by the number of phased elongation resulting in approximately equal filopodia lengths regardless of the substrate coating.We are now observing the movement of myosin-X lacking FERM domain using the same substrate coatings. At this meeting, we will compare these results with the above in detail.

Adaptive force transmission in amoeboid cell migration

The leading front of a cell can either protrude as an actin-free membrane bleb that is inflated by actomyosin-driven contractile forces, or as an actin-rich pseudopodium, a site where polymerizing actin filaments push out the membrane. Pushing filaments can only cause the membrane to protrude if the expanding actin network experiences a retrograde counter-force, which is usually provided by transmembrane receptors of the integrin family. Here we show that chemotactic dendritic cells mechanically adapt to the adhesive properties of their substrate by switching between integrin-mediated and integrin-independent locomotion. We found that on engaging the integrin-actin clutch, actin polymerization was entirely turned into protrusion, whereas on disengagement actin underwent slippage and retrograde flow. Remarkably, accelerated retrograde flow was balanced by an increased actin polymerization rate; therefore, cell shape and protrusion velocity remained constant on alternating substrates. Due to this adaptive response in polymerization dynamics, tracks of adhesive substrate did not dictate the path of the cells. Instead, directional guidance was exclusively provided by a soluble gradient of chemoattractant, which endowed these 'amoeboid' cells with extraordinary flexibility, enabling them to traverse almost every type of tissue.

Both actin and polyproline interactions of profilin-1 are required for migration, invasion and capillary morphogenesis of vascular endothelial cells

The objective of the present study was to evaluate how different ligand interactions of profilin-1 (Pfn1), an actin-binding protein that is upregulated during capillary morphogenesis of vascular endothelial cells (VEC), contribute to migration and capillary forming ability of VEC. We adopted a knockdown–knockin experimental system to stably express either fully functional form or mutants of Pfn1 that are impaired in binding to two of its major ligands, actin (H119E mutant) and proteins containing polyproline domains (H133S mutant), in a human dermal microvascular cell line (HmVEC) against near-null endogenous Pfn1 background. We found that silencing endogenous Pfn1 expression in HmVEC leads to slower random migration, reduced velocity of membrane protrusion and a significant impairment in matrigel-induced cord formation. Only re-expression of fully functional but not any of the two ligand-binding deficient mutants of Pfn1 rescues the above defects. We further show that loss of Pfn1 expression in VEC inhibits three-dimensional capillary morphogenesis, MMP2 secretion and ECM invasion. VEC invasion through ECM is also inhibited when actin and polyproline interactions of Pfn1 are disrupted. Together, these experimental data demonstrate that Pfn1 regulates VEC migration, invasion and capillary morphogenesis through its interaction with both actin and proline-rich ligands.

Pre-imbibition: Effects in the germination, seedling growth and content of caffeine in coffee seeds (Coffea arabica L.)

Coffee seeds ( Coffea arabica L.) contains from 1% to 2% of caffeine, a highly water-soluble alkaloid, which inhibits germination of several species. This work was undertaken to evaluate the effects of treatments of pre-imbibition and running water washing on physiological performance and caffeine content of coffee seeds, with the following treatments: pre-imbibition in water under stirring for 6, 12 and 24 hours, followed by fast washing in running water; washing in running water for 6, 12 and 24 hours; pre-imbibition in water for 6, 12, 24, 48, 72 and 96 hours, followed by fast washing in running water, and dry seeds witout parchment. The percentage of root protrusion, of normal seedlings at 30 days, of cotiledonary leaves at 45 days and of the root protrusion velocity index were evaluated. The contents of caffeine, aminoacids and total soluble sugars present in the imbibition solution, as well as the caffeine contents in seeds were also determinated. There were no significant differences in the root protrusion evaluation at fifteen days and the best treatments, evaluated by means of radical protrusion at seven days and of index of germination velocity, were pre-imbibited in water for 48, 72 and 96 hours. As to the percentage of seedlings with cotyledonary leaves opened at 45 days, the pre-imbibition treatments for both 24 and 48 hours provided the best physiological performances of seeds, and in these treatments there were significant reductions of caffeine content between 10% and 18%, respectively. Nevertheless, the detrimental action of caffeine on the physiological performance of coffee seeds, associated with the pre-imbibition treatments should be better investigated.