Arp2/3 complex interactions and actin network turnover in lamellipodia

Cell migration is initiated by lamellipodia—membrane-enclosed sheets of cytoplasm containing densely packed actin filament networks. Although the molecular details of network turnover remain obscure, recent work points towards key roles in filament nucleation for Arp2/3 complex and its activator WAVE complex. Here, we combine fluorescence recovery after photobleaching (FRAP) of different lamellipodial components with a new method of data analysis to shed light on the dynamics of actin assembly/disassembly. We show that Arp2/3 complex is incorporated into the network exclusively at the lamellipodium tip, like actin, at sites coincident with WAVE complex accumulation. Capping protein likewise showed a turnover similar to actin and Arp2/3 complex, but was confined to the tip. In contrast, cortactin—another prominent Arp2/3 complex regulator—and ADF/cofilin—previously implicated in driving both filament nucleation and disassembly—were rapidly exchanged throughout the lamellipodium. These results suggest that Arp2/3- and WAVE complex-driven actin filament nucleation at the lamellipodium tip is uncoupled from the activities of both cortactin and cofilin. Network turnover is additionally regulated by the spatially segregated activities of capping protein at the tip and cofilin throughout the mesh

F- and G-Actin Concentrations in Lamellipodia of Moving Cells

Cells protrude by polymerizing monomeric (G) into polymeric (F) actin at the tip of the lamellipodium. Actin filaments are depolymerized towards the rear of the lamellipodium in a treadmilling process, thereby supplementing a G-actin pool for a new round of polymerization. In this scenario the concentrations of F- and G-actin are principal parameters, but have hitherto not been directly determined. By comparing fluorescence intensities of bleached and unbleached regions of lamellipodia in B16-F1 mouse melanoma cells expressing EGFP-actin, before and after extraction with Triton X-100, we show that the ratio of F- to G-actin is 3.2+/−0.9. Using electron microscopy to determine the F-actin content, this ratio translates into F- and G-actin concentrations in lamellipodia of approximately 500 µM and 150 µM, respectively. The excess of G-actin, at several orders of magnitude above the critical concentrations at filament ends indicates that the polymerization rate is not limited by diffusion and is tightly controlled by polymerization/depolymerization modulators

Coordination of Membrane and Actin Cytoskeleton Dynamics during Filopodia Protrusion

Leading edge protrusion of migrating cells involves tightly coordinated changes in the plasma membrane and actin cytoskeleton. It remains unclear whether polymerizing actin filaments push and deform the membrane, or membrane deformation occurs independently and is subsequently stabilized by actin filaments. To address this question, we employed an ability of the membrane-binding I-BAR domain of IRSp53 to uncouple the membrane and actin dynamics and to induce filopodia in expressing cells. Using time-lapse imaging and electron microscopy of IRSp53-I-BAR-expressing B16F1 melanoma cells, we demonstrate that cells are not able to protrude or maintain durable long extensions without actin filaments in their interior, but I-BAR-dependent membrane deformation can create a small and transient space at filopodial tips that is subsequently filled with actin filaments. Moreover, the expressed I-BAR domain forms a submembranous coat that may structurally support these transient actin-free protrusions until they are further stabilized by the actin cytoskeleton. Actin filaments in the I-BAR-induced filopodia, in contrast to normal filopodia, do not have a uniform length, are less abundant, poorly bundled, and display erratic dynamics. Such unconventional structural organization and dynamics of actin in I-BAR-induced filopodia suggests that a typical bundle of parallel actin filaments is not necessary for generation and mechanical support of the highly asymmetric filopodial geometry. Together, our data suggest that actin filaments may not directly drive the protrusion, but only stabilize the space generated by the membrane deformation; yet, such stabilization is necessary for efficient protrusion

Comparative Dynamics of Retrograde Actin Flow and Focal Adhesions: Formation of Nascent Adhesions Triggers Transition from Fast to Slow Flow

Dynamic actin network at the leading edge of the cell is linked to the extracellular matrix through focal adhesions (FAs), and at the same time it undergoes retrograde flow with different dynamics in two distinct zones: the lamellipodium (peripheral zone of fast flow), and the lamellum (zone of slow flow located between the lamellipodium and the cell body). Cell migration involves expansion of both the lamellipodium and the lamellum, as well as formation of new FAs, but it is largely unknown how the position of the boundary between the two flow zones is defined, and how FAs and actin flow mutually influence each other. We investigated dynamic relationship between focal adhesions and the boundary between the two flow zones in spreading cells. Nascent FAs first appeared in the lamellipodium. Within seconds after the formation of new FAs, the rate of actin flow decreased locally, and the lamellipodium/lamellum boundary advanced towards the new FAs. Blocking fast actin flow with cytochalasin D resulted in rapid dissolution of nascent FAs. In the absence of FAs (spreading on poly-L-lysine-coated surfaces) retrograde flow was uniform and the velocity transition was not observed. We conclude that formation of FAs depends on actin dynamics, and in its turn, affects the dynamics of actin flow by triggering transition from fast to slow flow. Extension of the cell edge thus proceeds through a cycle of lamellipodium protrusion, formation of new FAs, advance of the lamellum, and protrusion of the lamellipodium from the new base

Bistability in the actin cortex

Multi-color fluorescence imaging experiments of wave forming Dictyostelium cells have revealed that actin waves separate two domains of the cell cortex that differ in their actin structure and phosphoinositide composition. We propose a bistable model of actin dynamics to account for these experimental observation. The model is based on the simplifying assumption that the actin cytoskeleton is composed of two distinct network types, a dendritic and a bundled network. The two structurally different states that were observed in experiments correspond to the stable fixed points in the bistable regime of this model. Each fixed point is dominated by one of the two network types. The experimentally observed actin waves can be considered as trigger waves that propagate transitions between the two stable fixed points

The use of Bayesian latent class cluster models to classify patterns of cognitive performance in healthy ageing

The main focus of this study is to illustrate the applicability of latent class analysis in the assessment of cognitive performance profiles during ageing. Principal component analysis (PCA) was used to detect main cognitive dimensions (based on the neurocognitive test variables) and Bayesian latent class analysis (LCA) models (without constraints) were used to explore patterns of cognitive performance among community-dwelling older individuals. Gender, age and number of school years were explored as variables. Three cognitive dimensions were identified: general cognition (MMSE), memory (MEM) and executive (EXEC) function. Based on these, three latent classes of cognitive performance profiles (LC1 to LC3) were identified among the older adults. These classes corresponded to stronger to weaker performance patterns (LC1>LC2>LC3) across all dimensions; each latent class denoted the same hierarchy in the proportion of males, age and number of school years. Bayesian LCA provided a powerful tool to explore cognitive typologies among healthy cognitive agers.The study is integrated in the "Maintaining health in old age through homeostasis (SWITCHBOX)" collaborative project funded by the European Commission FP7 initiative (grant HEALTH-F2-2010-259772). NS and JAP are main team members of the European consortium SWITCHBOX (http://www.switchbox-online.eu/). NCS is supported by a SwitchBox post-doctoral fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

The elementary events underlying force generation in neuronal lamellipodia

We have used optical tweezers to identify the elementary events underlying force generation in neuronal lamellipodia. When an optically trapped bead seals on the lamellipodium membrane, Brownian fluctuations decrease revealing the underlying elementary events. The distribution of bead velocities has long tails with frequent large positive and negative values associated to forward and backward jumps occurring in 0.1–0.2 ms with varying amplitudes up to 20 nm. Jump frequency and amplitude are reduced when actin turnover is slowed down by the addition of 25 nM Jasplakinolide. When myosin II is inhibited by the addition of 20 μM Blebbistatin, jump frequency is reduced but to a lesser extent than by Jasplainolide. These jumps constitute the elementary events underlying force generation