Protrusion Rate Research Articles

A Two-Dimensional Model of Leading Edge Protrusion and Retraction Driven by Excitable Actin Dynamics

Periodic patterns of protrusion and retraction have been observed along the leading edge of various cell types during spreading and motility, with many cells exhibiting wave-like propagation of protrusions along the cell membranes. Research has surged in this area as the reproducibility of these patterns offers opportunities to better quantify the regulatory mechanisms of actin polymerization at the leading edge. Previous works suggest possible roles for membrane curvature sensing proteins, actin filament nucleators, Rho signaling, myosin contraction, and severing in this process. To investigate cycles of protrusion and retraction we imaged the lamellipodia of XTC cells using Lifeact as a marker for actin, and analyzed the position of the leading edge using active contours. Constant retrograde flow indicated that protrusions and retractions were driven by fluctuations of the actin polymerization rate. We developed a 1D model of these actin dynamics as an excitable system in which a diffusive, autocatalytic activator causes free barbed end formation and actin polymerization; F-actin accumulation in turn inhibits further activator accumulation. According to the model, the leading edge protrusion rate spikes before the maximum of the free barbed end concentration. To gain mechanistic insight into how polymerization is converted into protrusion, we extended the model in 2D. F-actin dependent membrane motion and retrograde flow are both accounted for. We compare models of free barbed end generation to experiment using spatiotemporal correlations of leading edge velocity with concentrations of Arp2/3 complex, capping protein and VASP, as a function of distance from the leading edge. Our findings suggest that the Arp2/3 complex participates in an activation mechanism that includes additional diffusive components.

Intrinsic directionality of migrating vascular smooth muscle cells is regulated by NAD+ biosynthesis

Cell migration is central to tissue repair and regeneration but must proceed with precise directionality to be productive. Directional migration requires external cues but also depends on the extent to which cells can inherently maintain their direction of crawling. We report that the NAD(+) biosynthetic enzyme, nicotinamide phosphoribosyltransferase (Nampt/PBEF/visfatin), mediates directionally persistent migration of vascular smooth muscle cells (SMCs). Time-lapse microscopy of human SMCs subjected to Nampt inhibition revealed chaotic motility whereas SMCs transduced with the Nampt gene displayed highly linear migration paths. Ordered motility conferred by Nampt was associated with downsizing of the lamellipodium, reduced lamellipodium wandering around the cell perimeter, and increased lamellipodial protrusion rates. These protrusive and polarity-stabilizing effects also enabled spreading SMCs to undergo bipolar elongation to an extent not typically observed in vitro. Nampt was found to localize to lamellipodia and fluorescence recovery of Nampt-eGFP after photobleaching revealed microtubule-dependent transport of Nampt to the leading edge. In addition, Nampt was found to associate with, and activate, Cdc42, and Nampt-driven directional persistence and lamellipodium anchoring required Cdc42. We conclude that high-fidelity SMC motility is coordinated by a Nampt-Cdc42 axis that yields protrusive but small and anchored lamellipodia. This novel, NAD(+)-synthesis-dependent control over motility may be crucial for efficient repair and regeneration of the vasculature, and possibly other tissues.

Redundant Mechanisms for Stable Cell Locomotion Revealed by Minimal Models

Crawling of eukaryotic cells on flat surfaces is underlain by the protrusion of the actin network, the contractile activity of myosin II motors, and graded adhesion to the substrate regulated by complex biochemical networks. Some crawling cells, such as fish keratocytes, maintain a roughly constant shape and velocity. Here we use moving-boundary simulations to explore four different minimal mechanisms for cell locomotion: 1), a biophysical model for myosin contraction-driven motility; 2), a G-actin transport-limited motility model; 3), a simple model for Rac/Rho-regulated motility; and 4), a model that assumes that microtubule-based transport of vesicles to the leading edge limits the rate of protrusion. We show that all of these models, alone or in combination, are sufficient to produce half-moon steady shapes and movements that are characteristic of keratocytes, suggesting that these mechanisms may serve redundant and complementary roles in driving cell motility. Moving-boundary simulations demonstrate local and global stability of the motile cell shapes and make testable predictions regarding the dependence of shape and speed on mechanical and biochemical parameters. The models shed light on the roles of membrane-mediated area conservation and the coupling of mechanical and biochemical mechanisms in stabilizing motile cells.

An Adhesion-Dependent Switch between Mechanisms That Determine Motile Cell Shape

Keratocytes are fast-moving cells in which adhesion dynamics are tightly coupled to the actin polymerization motor that drives migration, resulting in highly coordinated cell movement. We have found that modifying the adhesive properties of the underlying substrate has a dramatic effect on keratocyte morphology. Cells crawling at intermediate adhesion strengths resembled stereotypical keratocytes, characterized by a broad, fan-shaped lamellipodium, clearly defined leading and trailing edges, and persistent rates of protrusion and retraction. Cells at low adhesion strength were small and round with highly variable protrusion and retraction rates, and cells at high adhesion strength were large and asymmetrical and, strikingly, exhibited traveling waves of protrusion. To elucidate the mechanisms by which adhesion strength determines cell behavior, we examined the organization of adhesions, myosin II, and the actin network in keratocytes migrating on substrates with different adhesion strengths. On the whole, our results are consistent with a quantitative physical model in which keratocyte shape and migratory behavior emerge from the self-organization of actin, adhesions, and myosin, and quantitative changes in either adhesion strength or myosin contraction can switch keratocytes among qualitatively distinct migration regimes.

An Autocatalytic Activator-Inhibitor Model of Actin Polymerization at the Leading Edge of Fibroblast Lamellipodia

Crawling animal cells are driven by lamellipodia -- thin, dynamic, sheet-like projections consisting of a dense network of branched actin filaments. Arp2/3 complex mediated actin polymerization near the leading edge drives protrusion required for cellular motility. Actin filaments polymerize near the leading edge and undergo retrograde flow towards the main body of the cell. Both random fluctuations and travelling waves of protrusion and retraction have been observed during cell spreading. To characterize the protrusion rates of XTC fibroblasts we use active contours to track the leading edge of cells. We then calculate the correlation between local actin and Arp2/3 complex accumulation and cell protrusion rate as measured from conventional fluorescent microscopy. We also use quantitative fluorescent speckle microscopy, which allows us to track the dynamics of individual proteins within the actin network and measure quantities such as the retrograde flow rate. We present a noise-driven model of these actin-mediated protrusion events in which a diffusive, autocatalytic activator mediates actin polymerization, while actin saturation in turn inhibits further activator accumulation. Using analytic theory we find that our model captures both excitable and oscillatory behaviors, and can be tuned between the two behaviors by adjusting model parameters. Using computer simulation we are able to produce patterns of actin polymerization that are quantitatively similar to those observed along the leading edge in live cell experiments.

Lumbar Disk Protrusion Rates of Symptomatic Patients Using Magnetic Resonance Imaging

Objective The purpose of this study was to determine the rate of disk protrusions detected via magnetic resonance imaging (MRI) in patients symptomatic for spine pain, radiculopathy, or other spine-related pain. Methods A retrospective review of 1983 MRI scans was performed over a 2-year period on 1486 patients, each of whom was symptomatic for spine pain, radiculopathy, or other noncancer, spine-related pain. Of these patients, 761 were scanned in the recumbent position using low-field (0.3 T, Airis II, Hitachi, Twinsburg, Ohio) MRI, and 725 were scanned in an upright, sitting position using mid-field (0.6 T) open Upright MRI (Fonar, Melville, NY). In total, 986 serial scans were performed on patients in the recumbent position and 997 serial scans on patients in the weight-bearing position. Results One or more disk protrusions were identified in 73.3% of scans performed in the sitting position and in 50.1% of scans performed in the recumbent position. Most disk protrusions occurred at L5-S1 (52.3% and 29.8%), L4-L5 (42.6% and 26.7%), and L3-L4 (26.7% and 13.1%) in upright and recumbent positions, respectively. Conclusions The disk protrusion rate in this group of patients ranged between 50.1% (recumbent) and 73.3% (weight-bearing). These rates are higher than rates reported in the medical literature for asymptomatic patients, a finding that supports the decision to further evaluate patients with persistent spine-related pain.

αvβ3 integrin spatially regulates VASP and RIAM to control adhesion dynamics and migration

Integrins are fundamental to the control of protrusion and motility in adherent cells. However, the mechanisms by which specific members of this receptor family cooperate in signaling to cytoskeletal and adhesion dynamics are poorly understood. Here, we show that the loss of beta3 integrin in fibroblasts results in enhanced focal adhesion turnover and migration speed but impaired directional motility on both 2D and 3D matrices. These motility defects are coupled with an increased rate of actin-based protrusion. Analysis of downstream signaling events reveals that loss of beta3 integrin results in a loss of protein kinase A-dependent phosphorylation of the actin regulatory protein vasodilator-stimulated phosphoprotein (VASP). Dephosphorylated VASP in beta3-null cells is preferentially associated with Rap1-GTP-interacting adaptor molecule (RIAM) both in vitro and in vivo, which leads to enhanced formation of a VASP-RIAM complex at focal adhesions and subsequent increased binding of talin to beta1 integrin. These data demonstrate a novel mechanism by which alphavbeta3 integrin acts to locally suppress beta1 integrin activation and regulate protrusion, adhesion dynamics, and persistent migration.

Proteomics reveal tissue‐specific features of the cress (Lepidium sativum L.) endosperm cap proteome and its hormone‐induced changes during seed germination

Mature angiosperm seeds consist of an embryo surrounded by the endosperm and the testa. The endosperm cap that covers the radicle plays a regulatory role during germination and is a major target of abscisic acid-induced inhibition of germination. Cress (Lepidium sativum) is a close relative of the model plant Arabidopsis thaliana (Arabidopsis). Cress seeds offer the unique possibility of performing tissue-specific proteomics due to their larger size while benefiting the genomic tools available for Arabidopsis. This work provides the first description of endosperm cap proteomics during seed germination. An analysis of the proteome of the cress endosperm cap at key stages during germination and after radicle protrusion in the presence and absence of abscisic acid led to the identification of 144 proteins, which were clustered by the changes in their abundances and categorized by function. Proteins with a function in energy production, protein stability and stress response were overrepresented among the identified endosperm cap proteins. This strongly suggests that the cress endosperm cap is not a storage tissue as the cereal endosperm but a metabolically very active tissue regulating the rate of radicle protrusion.

Quantitative Analysis of Cell Edge Dynamics and Cell Shape in Non-Polarized Fish Epidermal Keratocytes

Cell migration requires the coordination of several processes such as leading edge protrusion, adhesion formation and disassembly, and trailing edge retraction. The quantitative analysis concerning the correlation between these processes will be useful to understand the coordination mechanism. A major difficulty in the quantitative analysis stems from the complex morphology of migrating cells. To cope with this difficulty, we selected the non-polarized fish epidermal keratocytes as a simplified experimental system that includes basal migratory mechanisms. We acquired a mixture of non-polarized stationary keratocytes and polarized highly motile keratocytes by disaggregating the large epidermal sheets. The time-lapse micrographs of non-polarized keratocytes were used to analyze cell edge dynamics and cell peripheral shape. We adopted the protrusion and retraction rate and cell peripheral curvature as quantitative parameters. Non-polarized keratocytes did not exhibit net translocation, however, active protrusion and retraction were observed around the cell periphery. Protrusion rate was negatively correlated with the cell peripheral curvature. In contrast, retraction rate was positively correlated with the cell peripheral curvature. The plot of protrusion and retraction rates over the entire cell periphery showed that protrusion and retraction waves were traveling laterally in both directions along the cell periphery. The lateral traveling velocity of each wave was constant. The wave persistence varied from 10 s to 100 s. These results indicate that the cell has the positive feedback mechanism maintaining stable protrusion and retraction and that the rate of protrusion and retraction is related to the cell peripheral shape. Quantitative analysis together with theoretical and molecular biological studies will shed light on the mechanism of cell migration.

Redundant Mechanisms for Stable Cell Locomotion Revealed by Minimal Models

Crawling of eukaryotic cells on flat surfaces is underlain by the dynamics of the actin network and graded adhesion to the substrate and is regulated via a complex biochemical network. Some crawling cells maintain roughly constant shape and velocity. The paradigm of this stable crawling is the fish keratocyte, a rapidly moving cell that maintains a half-moon shape while translocating. Here we use moving boundary simulations to explore 4 different, minimal mechanisms for cell locomotion and show that all of these are sufficient to produce steady shapes and movements with resulting features that resemble the keratocyte morphology. We begin by considering a diffusion-limited actin model where G-actin transport to the leading edge controls the rate of protrusion of the leading edge. The second model treats actin contraction by myosin as the primary determinant of shape and speed. Simulations show that this model is able to produce very realistic keratocyte-like shape and also reproduces the bistability that is observed in keratocyte fragments. We next consider a simple biochemical regulation model that has been proposed for the polarization of the Rac/Rho system. Here a high concentration of Rac at the front of the cell stimulates actin polymerization, and Rho at the rear of the cell induces contraction of myosin. Interestingly, volume conservation is not required for this model to work, yet the model behaves very differently if the cell has fixed volume than when the volume is self-regulating. The final model assumes that microtubule-based transport of vesicles to the leading edge limits the rate of protrusion. As all of these models are able to produce steady crawling locomotion, it suggests that these mechanisms may serve redundant roles in driving cell motility.

Erectile Implants in Female-to-Male Transsexuals: Our Experience in 129 Patients

BackgroundThe combination of a neourethra and erection prosthesis in a single neophallus in the female-to-male transsexual remains a challenge. No good data are available on this subject. ObjectiveTo report the outcome in 129 female-to-male transsexuals with a neophallus after the implantation of an erectile prosthesis. Design, setting, and participantsFrom March 1996 until October 2007, 129 female-to-male transsexuals with a neophallus underwent the implantation of an erectile prosthesis. The mean follow-up was 30.2 mo (range: 0–132 mo). InterventionA Dynaflex prosthesis was implanted initially in 9 patients, a three-piece hydraulic device (AMS CX or AMS CXM) in 50 patients, and a CX Inhibizone, Ambicor, and Coloplast/Mentor prosthesis in 17, 47, and 6 patients, respectively. MeasurementsData on outcome in these patients were retrospectively evaluated. Results and limitationsOf 129 patients, 76 patients (58.9%) still have their original implant in place. Fifty-three patients (41.1%) needed to undergo either removal or revision of the prosthesis due to infection, erosion, dysfunction, or leak. Forty-one patients underwent a replacement of the prosthesis, nine needed a second revision, five needed a third revision, and one patient needed a fourth revision of prosthesis. Malposition of prosthesis was corrected by surgical repositioning so that removal or revision could be avoided. Of 185 prostheses used in 129 patients, 108 (58.4%) still remain in place, with a total infection rate of 11.9%, a total protrusion rate of 8.1%, a total prosthesis leak rate of 9.2%, a total dysfunction rate of 13%, and a total malposition rate of 14.6%.The period of follow-up in the more recent types of prostheses (Ambicor, Coloplast/Mentor) is much shorter; therefore, comparison with earlier types is difficult to make. ConclusionsDespite high complication rates, implantation of a hydraulic erectile prosthesis remains the best option for achieving the possibility of sexual intercourse in female-to-male transsexuals.

Cortical control of appropriate tongue protrusion during licking in cats—Increase in regional cerebral blood flow (rCBF) of the contralateral area P and in tongue protrusion after the unilateral area P lesion

Adequate tongue protrusion may be regulated by cat bilateral area P (the motor cortex for jaw and tongue movements) (Hiraba and Sato, Somatosens Mot Res 2005b;22:183–192). The ICMS (intracortical microstimulation) in the unilateral area P evoked motor effects of tongue protrusion without deviation (Hiraba and Sato, Somatosens Mot Res 2004;23:1–12), and cats with the unilateral lesion of area P showed abnormal tongue protrusion without deviation during licking (Hiraba and Sato, Somatosens Mot Res 2005b;22:183–192). Further, the measurements of the regional cerebral blood flow (rCBF) in the bilateral jaw and tongue motor cortical areas were shown to have the same activity rate during the lateral licking (Hiraba and Sato, Somatosens Mot Res 2005c;22:307–317). We assumed from these results that cortical control for tongue protrusion was executed by networks between the bilateral area P including inhibitory interneurons. We prepared the measurable cats of the rCBF in the contralateral side after the unilateral area P lesion. Changes in the rates of rCBF and tongue protrusion during licking were examined over a long time course of about 1–2 months after the unilateral area P lesion. All cats after the unilateral area P lesion showed increased rate (double or triple in comparison with the normal ones) of rCBF of the contralateral area P in the early (0–20 days) phase. On the other hand, increased rates of tongue protrusion were about 120% in the early phase, and about 180% in the middle (21–35 days) and late (36–last days) phases. The results support the organization of networks between bilateral area P including the inhibitory interneurons.

Loss of profilin‐1 expression enhances breast cancer cell motility by Ena/VASP proteins

We previously showed that silencing profilin-1 (Pfn1) expression increases breast cancer cell motility, but the underlying mechanisms have not been explored. Herein, we demonstrate that loss of Pfn1 expression leads to slower but more stable lamellipodial protrusion thereby enhancing the net protrusion rate and the overall motility of MDA-MB-231 breast cancer cells. Interestingly, MDA-MB-231 cells showed dramatic enrichment of VASP at their leading edge when Pfn1 expression was downregulated and this observation was also reproducible in other cell types including human mammary epithelial cells and vascular endothelial cells. We further demonstrate that Pfn1 downregulation results in a hyper-motile phenotype of MDA-MB-231 cells in an Ena/VASP-dependent mechanism. Pfn1-depleted cells display a strong colocalization of VASP with lamellipodin (Lpd--a PI(3,4)P(2)-binding protein that has been previously implicated in lamellipodial targeting of Ena/VASP) at the leading edge. Finally, inhibition of PI3-kinase (important for generation of PI(3,4)P(2)) delocalizes VASP from the leading edge. This observation is consistent with a possible involvement of Lpd in enhanced membrane recruitment of VASP that results from loss of Pfn1 expression. Our findings for the first time highlight a possible mechanism of how reduced expression of a pro-migratory molecule like Pfn1 could actually promote motility of breast cancer cells.

Transanal protrusion of intussusception in infants is associated with high morbidity and mortality

Although anal protrusion of intussusception in infants is well recognised, it is rarely reported and confusion with rectal prolapse often results in delayed diagnosis and treatment. This report highlights the problems of diagnosis and the morbidity and mortality associated with this condition. A retrospective case series of five infants presenting to Ahmadu Bello University Teaching Hospital (ABUTH), Zaria, Nigeria with anal protrusion of intussusception over a period of 5 years. During that time, 17 children were treated for intussusception at ABUTH. The anal protrusion rate of intussusception was 29%. The five infants were three girls and two boys aged 4-18 months (median 8). The duration of symptoms was between 6 and 28 days (median 21). The features were mainly protruding anal mass, diarrhoea and vomiting. Abdominal pain and passage of bloody stools occurred late. There was delay in referral and treatment owing to misdiagnosis as rectal prolapse. The intussusception was ileocolic in four patients and in one the type could not be ascertained before death. Two patients had perforation of the involved intestine and another had gangrene of the intestine, necessitating intestinal resection. In one patient, there was no bowel compromise and only open reduction was necessary. Two patients died from overwhelming infection, one before surgery and another after surgery. The risk of morbidity and mortality in anal protruding intussusception is high. As early features might not be typical of intussusception, a high index of suspicion is necessary to avoid confusion with rectal prolapse so as not to delay diagnosis and treatment.

Actin and α-actinin orchestrate the assembly and maturation of nascent adhesions in a myosin II motor-independent manner

Using two-colour imaging and high resolution TIRF microscopy, we investigated the assembly and maturation of nascent adhesions in migrating cells. We show that nascent adhesions assemble and are stable within the lamellipodium. The assembly is independent of myosin II but its rate is proportional to the protrusion rate and requires actin polymerization. At the lamellipodium back, the nascent adhesions either disassemble or mature through growth and elongation. Maturation occurs along an alpha-actinin-actin template that elongates centripetally from nascent adhesions. Alpha-Actinin mediates the formation of the template and organization of adhesions associated with actin filaments, suggesting that actin crosslinking has a major role in this process. Adhesion maturation also requires myosin II. Rescue of a myosin IIA knockdown with an actin-bound but motor-inhibited mutant of myosin IIA shows that the actin crosslinking function of myosin II mediates initial adhesion maturation. From these studies, we have developed a model for adhesion assembly that clarifies the relative contributions of myosin II and actin polymerization and organization.

Role of aminergic (serotonin and dopamine) systems in the embryogenesis and different embryonic behaviors of the pond snail, Lymnaea stagnalis

A detailed biochemical and pharmacological analysis of the dopaminergic (DAergic) and serotonergic (5-HTergic) systems was performed during the embryogenesis of Lymnaea stagnalis, to monitor their role in development and different behaviors. The dopamine (DA) level and the synthesizing decarboxylase enzyme activity showed a continuous increase, whereas the serotonin (5-HT) concentration remained low until late postmetamorphic development, when they all showed a rapid and significant increase. Application of monoamine precursors increased, whereas enzyme inhibitors and neurotoxins reduced monoamine levels; all treatments resulting in a prolongation of embryogenesis. Following, p-chlorphenylalanine (pCPA) and 3-hydroxybenzylhydrazine (Nsd-1015) treatments, no 5-HT immunoreactivity could be detected in the embryonic nervous system. These findings suggest that changes of monoamine levels in either (negative or positive) direction cause slowing of embryogenesis. Embryonic rotation and radula protrusion rate was enhanced following both serotonin and dopamine application, whereas frequency of gliding was increased by serotonin treatment. These results clearly indicate the involvement of 5-HT and DA in the regulation of a broad range of embryonic behaviors. Pharmacological characterization of a 5-HT receptor associated with the L. stagnalis embryonic behaviors studied revealed that a mammalian 5-HT 1-like receptor type is involved in the 5-HTergic regulation of locomotion activity.

Perioperative Complications and Early Follow-up with 100 TVT-SECUR Procedures

Our objective was to evaluate the complications and early follow-up of the tension-free vaginal tape (TVT)-SECUR, a new minimally invasive anti-incontinence operative procedure. A prospective, observational, and consecutive patient series was conducted. Perioperative and 12-month postoperative data were prospectively collected for the first 50 patients against the next consecutive 50 patients, among which TVT-SECUR specific surgical measurements were adopted (Canadian Task Force classification 2). In private hospital operative theatres, the TVT-SECUR operation was performed. Patients with urodynamically proved stress urinary incontinence were enrolled in this study after detailed informed consent was given. The TVT-SECUR, in the hammock shape to mimic the TVT-obturator placement, yet with no skin incisions, required neither bladder catheterization nor intraoperative diagnostic cystoscopy. The clinical and surgical data of 100 consecutive patients with TVT-SECUR were collected prospectively. Two patients had urinary obstructions and needed surgical tape-tension relief. One patient had a 50 mL paravesical self-remitting hematoma. At the first-month postoperative follow-up appointment, the objective therapeutic failure rate for the TVT-SECUR procedure among the 50 patients was 20.0% (10 patients). But when the tape was placed close to the urethra with no space allowed in between, the failure rate in the second patient group went down to 8.0% (4 patients); yet no further postoperative bladder outlet obstruction was diagnosed. Four (8.0%) patients in the first group had vaginal wall penetration with the inserters, requiring withdrawal, reinsertion, and vaginal wall repair. This was avoided with the second patient group by facilitating the inserters' introduction by widening the submucosal tunnel to 12 mm. Six (12.0%) other patients in the first group needed postoperative trimming of a vaginally extruded tape segment, performed in the office with satisfactory results. This problem was addressed later by making the submucosal dissection deeper to avoid intimate proximity of the tape with the vaginal mucosa. Consequently the tape protrusion rate was reduced to 8% (4 patients). Five (10.0%) patients in the first group had unintended tape removal at the time of inserter removal, necessitating the use of a second TVT-SECUR. This was addressed by meticulous detachment of the inserter before its withdrawal, after which no further unintended tape displacements were recorded. No clinical signs for bowel, bladder, or urethral injuries; intraoperative bleeding; or postoperative infections were evident. Telephone interview at the end of 12 months postoperatively was completed with 44 (88.0%) of the first patient group and 46 (92%) of the second patient group. In all, 39 (88.6%) and 43 (93.5%) of the telephone-interviewed patients of the first and second groups, respectively, reported objective urinary continence. The TVT-SECUR, a new midurethral sling, was associated with early safety and efficacy problems. These were identified and rectified, to make the TVT-SECUR a safe and effective anti-incontinence procedure. Operative complications associated with the TVT, such as bladder penetration and postoperative outlet obstruction, and TVT-obturator complications, such as postoperative thigh pain and bladder outlet obstruction, may be reduced with the TVT-SECUR. The first 100 operations' cumulative data analysis yielded some insights, including the necessity of meticulous and proper dissection before placement of the tape and the need for applying minimal extra tension to the tape. However, long-term comparative data collection will be required to draw solid conclusions regarding the appropriate position of this operative technique within the spectrum of anti-incontinence operations.

Differentially oriented populations of actin filaments generated in lamellipodia collaborate in pushing and pausing at the cell front

Eukaryotic cells advance in phases of protrusion, pause and withdrawal. Protrusion occurs in lamellipodia, which are composed of diagonal networks of actin filaments, and withdrawal terminates with the formation of actin bundles parallel to the cell edge. Using correlated live-cell imaging and electron microscopy, we have shown that actin filaments in protruding lamellipodia subtend angles from 15-90 degrees to the front, and that transitions from protrusion to pause are associated with a proportional increase in filaments oriented more parallel to the cell edge. Microspike bundles of actin filaments also showed a wide angular distribution and correspondingly variable bilateral polymerization rates along the cell front. We propose that the angular shift of filaments in lamellipodia serves in adapting to slower protrusion rates while maintaining the filament densities required for structural support; further, we suggest that single filaments and microspike bundles contribute to the construction of the lamella behind and to the formation of the cell edge when protrusion ceases. Our findings provide an explanation for the variable turnover dynamics of actin filaments in lamellipodia observed by fluorescence speckle microscopy and are inconsistent with a current model of lamellipodia structure that features actin filaments branching at 70 degrees in a dendritic array.

Modulation of lamellipodial structure and dynamics by NO-dependent phosphorylation of VASP Ser239

The initial step in directed cell movement is lamellipodial protrusion, an action driven by actin polymerization. Enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) family proteins are key regulators of this actin polymerization and can control lamellipodial protrusion rate. Ena/VASP proteins are substrates for modification by cyclic-nucleotide-dependent protein kinases at a number of sites. Phosphorylation of Ser239 of VASP in vitro inhibits its anti-capping and filament-bundling activity but the effects of this modification on lamellipodial structure and function are unknown. To examine the functional effects of this modification in living cells, we studied VASP phosphorylation at Ser239 by nitric oxide (NO) stimulation of cGMP-dependent protein kinase. Using live cell imaging of primary cells transfected with GFP-VASP constructs, we found that NO produced rapid retraction of lamellipodia together with cell rounding that was dependent on guanylate cyclase and type II cGMP-dependent protein kinase. In cells expressing a mutant VASP (Ser239Ala) lacking the site preferentially phosphorylated by this kinase, NO had no effect. Phosphorylation of Ser239 of VASP results in loss of lamellipodial protrusions and cell rounding, and is a powerful means of controlling directed actin polymerization within lamellipodia.

Activity-Dependent PSD Formation and Stabilization of Newly Formed Spines in Hippocampal Slice Cultures

Development and remodeling of synaptic networks occurs through a continuous turnover of dendritic spines. However, the mechanisms that regulate the formation and stabilization of newly formed spines remain poorly understood. Here, we applied repetitive confocal imaging to hippocampal slice cultures to address these issues. We find that, although the turnover rate of protrusions progressively decreased during development, the process of stabilization of new spines remained comparable both in terms of time course and low level of efficacy. Irrespective of the developmental stage, most new protrusions were quickly eliminated, in particular filopodia, which only occasionally lead to the formation of stable dendritic spines. We also found that the stabilization of new protrusions was determined within a critical period of 24 h and that this coincided with an enlargement of the spine head and the expression of tagged PSD-95. Blockade of postsynaptic AMPA and NMDA receptors significantly reduced the capacity of new spines to express tagged PSD-95 and decreased their probability to be stabilized. These results suggest a model in which synaptic development is associated with an extensive, nonspecific growth of protrusions followed by stabilization of a few of them through a mechanism that involves activity-driven formation of a postsynaptic density.