Arp2/3 complex reconstructions reveal insights into the pathway of actin branch formation

The actin-related protein (Arp)2/3 complex interacts with nucleation-promoting factors (NPFs), pre-existing actin filaments and actin monomers to initiate the formation of new actin branches. NPF binding induces conformational changes in Arp2/3, but the exact nature of these changes and how the various components interact was not understood until now. By using electron microscopy and single-particle image analysis to generate three-dimensional reconstructions at ~2 nm resolution, Xu et al. have now uncovered two distinct conformations of the Arp2/3 complex in the presence or absence of three different NPFs — N-WASp, SCAR-VCA and cortactin. Three-dimensional reconstructions of NPF-free Arp2/3 complex were not significantly different in conformation from those of Arp2/3 complex crystal structures, indicating that this conformation represents the inactive state. In contrast, binding of NPFs induces distinct structural changes in Arp2/3 complex that are independent of the specific NPF bound. The most striking of these changes is the rearrangement of the Arp2 and Arp3 subunits into a geometry resembling a short-pitch actin-filament dimer, similar to that observed in the fully assembled branch junction. Interestingly, the NPF-bound Arp2/3 complex is incompatible with a required subsequent step for completion of branch formation: all reconstructions showed the NPFs attached at the pointed ends of Arp2 and Arp3, partially occluding the site where Arp2/3 complex binds pre-existing actin filaments. Distance constraints generated by fluorescence resonance energy transfer analysis confirmed the presence of NPFs at this location. Thus, the results of this study extend our knowledge of the pathway of Arp2/3-mediated branch formation in two ways: (1) NPF binding triggers a conformational change in Arp2/3 complex that brings Arp2 and Arp3 into a filament-like arrangement, forming a scaffold primed for nucleation of the new, branched filament; (2) additional changes are required to facilitate tight binding of Arp2/3 complex to pre-existing actin filaments, most likely involving the binding of a second NPF at a different site. The intricate choreography of these steps ensures a tight spatio-temporal regulation of actin-branch initiation. 

• Xu XP, Rouiller I, Slaughter BD, Egile C, Kim E, Unruh JR, Fan X, Pollard TD, Li R, Hanein D, Volkmann N. Three-dimensional reconstructions of Arp2/3 complex with bound nucleation promoting factors. EMBO J 2011; Sep 20. [Epub ahead of print] PubMed

FRET–FLIM analysis: time for a different approach?

Changes in biosensor fluorescence intensity or lifetime can theoretically be used to detect and quantitate Förster resonance energy transfer (FRET; also known as fluorescence resonance energy transfer) as a function of time and space. Among the detection methods for FRET, intensity-based ratiometric analysis displays several advantages, but is highly sensitive to fluorescence artifacts. Although fluorescence lifetime imaging microscopy (FLIM) provides a viable alternative, determining the contribution of individual fluorescent species among a mixture (which occurs in any FRET experiment) cannot easily be achieved by current analysis of FLIM data in the time domain, which requires fitting the average fluorescence decay in each pixel using a multi-exponential model. Hinde et al. have, however, presented fit-less phasor approach to the analysis of FLIM data and showed that this method of FRET detection can determine the spatiotemporal dynamics of biosensors independent of other cellular sources of fluorescence. The authors successfully applied this approach — which transforms each molecular species into a two-dimensional coordinate system — to the measurement of FRET activity of a Rac1 dual chain biosensor and two RhoA single chain biosensors, and demonstrated that it can quantify the local concentration of the free and bound state of the former, and the low and high FRET species of the latter, in each pixel of an image. The resulting map of the activity of these GTPases agrees with their localization during cell migration as reported in the literature, confirming the validity of this method. Moreover, Hinde et al. showed that phasor analysis can be used to characterize the physical properties that are intrinsic to the design of each biosensor, which should facilitate the more optimal design of biosensors during their development.

• Hinde E, Digman MA, Welch C, Hahn KM, Gratton E. Biosensor Förster resonance energy transfer detection by the phasor approach to fluorescence lifetime imaging microscopy. Microsc Res Tech. 2011 [Epub ahead of print] PubMed

Doing the uncaging two-step

The use of photolabile caging groups has been extended to facilitate precise spatial and temporal control over phosphorylation events, but current methods enable only one caged phosphopeptide to be assessed in any one experiment. Barbara Imperiali’s group has now developed a strategy to sequentially uncage two different phosphopeptides in one system by exploiting the distinct photophysical properties of two different caging groups — 1-(2-nitrophenol)ethyl (NPE) and [7-(diethylamino)coumarin-4-yl]-methyl (DEACM). Having previously generated NPE-caged phosphorylated serine, threonine and tyrosine, the group successfully synthesized DEACM-caged phosphorylated amino acid building blocks for Fmoc-based solid-phase peptide synthesis to enable their efficient incorporation into peptides and proteins. Notably, the DEACM chromophore shows maximal absorbance at 390 nm, whereas the NPE group absorbs minimally above 380 nm, theoretically enabling the selective release of both groups at different wavelengths. The authors then carried out in vitro studies to investigate the selective uncaging of a DEACM-caged phosphothreonine substrate in the presence of an NPE-caged phosphoserine inhibitor of Wip1, a phosphatase that indirectly suppresses the activity of p53. Irradiation of both peptides at 420 nm successfully uncaged the DEACM group, releasing the phosphothreonine substrate; subsequent irradiation at 365 nm uncaged the NPE group to release the phosphoserine inhibitor. In a Wip1 phosphatase assay, irradiation at 420 nm initiated phosphatase activity by uncaging the DEACM-caged substrate phosphothreonine, and subsequent exposure to 365 nm inhibited phosphatase activity by releasing the NPE-caged Wip1 inhibitor. The authors envisage that this wavelength-selective approach to controlling two different processes could be used in cell migration studies — for example, to shed light on the role of sequential phosphorylation of paxillin, or to investigate the influence of phosphorylation of regulatory proteins on the function of Rac.

• Goguen BN, Aemissegger A, Imperiali B. Sequential activation and deactivation of protein function using spectrally differentiated caged phosphoamino acids. J Am Chem Soc. 2011 133(29):11038-11041 PubMed

Taking a two-tiered approach to optimize scaffold colonization

The use of bone-marrow-derived multipotent stromal cells (MSCs) in conjunction with artificial scaffolds offers considerable potential for bone tissue engineering, but MSCs must effectively migrate beyond their seeding site in order to colonize the scaffold. To gain insight into characteristics of human telomerase reverse-transcriptase immortalized (hTERT) MSC migration, Wu et al. tracked hTERT MSCs by time-lapse imaging on varying concentrations of fibronectin, vitronectin or collagen-I adsorbed onto a copoly(methylmethacrylate)-grafted-poly(ethylene oxide) polymer surface in the presence of tethered epidermal growth factor (EGF), thereby providing extracellular matrix and growth factor signals. Speed, persistence, and the product of these two parameters, the mean free path (MFP), were lower, but more consistent, on collagen than on the other substrates, which both strongly influenced migration behaviors depending on their concentration. Wu et al. then measured the activities of four key signaling proteins downstream of EGF and ECM signals — the EGF receptor, extracellular signal-regulated kinase (ERK), Akt and focal adhesion kinase — to investigate their ability to affect migration speed or persistence. Unsurprisingly (given that migration requires the integration of several pathways), the activity of none of the proteins conferred significant predictive capacity for migratory behavior. The authors therefore used decision tree modeling, previously successfully applied to predictive understanding of fibroblast migration, to generate the non-intuitive hypothesis that decreasing ERK activity would increase MFP by increasing persistence. To test this experimentally, Wuet al. used U0126 to inhibit the kinase upstream of ERK in MSCs seeded on the copolymer surface with 3 μg/ml adsorbed collagen in the presence of tEGF. This partial inhibition of ERK significantly increased the persistence time and, therefore, the MFP. In the context of a biomaterials scaffold, an increased MFP might confer increased colonization vigor, so Wu et al. have successfully demonstrated the ability to optimize MSC migration using a two-tiered approach: ‘coarse-graining’ materials in conjunction with ‘fine-tuning’ signaling molecules.

• Wu S, Wells A, Griffith LG, Lauffenburger DA. Controlling multipotent stromal cell migration by integrating “coarse-graining” materials and “fine-tuning” small molecules via decision tree signal-response modeling. Biomaterials. 2011; 2(30):7524-31. PubMed

Sensing a change: new Cdc42 probe

Rho GTPases function as molecular switches, alternating between an inactive, GDP-bound state and an active, GTP-bound state, which binds downstream effectors. Existing methods for assessing endogenous Cdc42 activity make use of the Cdc42/Rac-interactive binding (CRIB) domain of one such effector, Wiskott–Aldrich syndrome protein (WASP), but by exploiting the properties of the fluorophore 4-N,N-dimethylamino-1,8-naphthalimide (4-DMN), Barbara Imperiali and colleagues have developed a sensor that offers considerable advantages over current approaches. The 4-DMN solvatochromic fluorophore is ideally suited for indicating a binding event and shows low levels of fluorescence in its unbound, water-exposed state but demonstrates marked fluorescence increases in a hydrophobic environment such as that at a protein-binding interface. Imperiali’s group incorporated 4-DMN into the CRIB domain of WASP (residues 230–277) by cysteine labeling or protein semisynthesis to generate a panel of five sensors. They then assessed the fluorescent properties of the sensor derivatives towards Cdc42 by incubating each of them with purified GDP-bound or GTP-γS-bound Cdc42. All sensors showed little, if any, background fluorescence in the presence of inactive GDP-bound Cdc42, but dramatic increases in fluorescence occurred in the presence of Cdc42 containing the non-hydrolyzable GTP-γS analog. One derivative, with a longer linker between the fluorophore and the body of the sensor, exhibited a 32-fold increase in fluorescence intensity upon Cdc42 activation. In measurements of real-time activity, this probe easily monitored intrinsic nucleotide exchange and hydrolysis, demonstrating further potential to assess the influence of Cdc42 regulatory proteins or even small-molecule modulators in a high-throughput format. Combined, these properties make the 4-DMN-based Cdc42 sensor a powerful tool for the study of the activity of this small GTPase.

• Goguen BN, Loving GS, Imperiali B. Development of a fluorogenic sensor for activated Cdc42. Bioorg Med Chem Lett. 2011 Apr 20. [Epub ahead of print] PubMed

Movement model takes shape

The balance between cell–substrate adhesion and myosin contraction is known to influence the speed of migrating cells, but the extent to which these elements contribute to determining the shape of motile cells is less apparent. Reporting in PLoS Biology, Barnhart et al. observed that adhesion strength had a modest, but biphasic, effect on migration speed in fish epithelial keratocytes: cells migrated faster at intermediate adhesion strengths than at low or high adhesion strengths. By contrast, keratocyte shape strongly depended on adhesion strength, resulting in small, round cells showing highly variable rates of protrusion and retraction at low strengths, fan-shaped cells on surfaces of intermediate adhesion strength, and large cells with traveling waves of protrusion at high strength. To investigate how changes in adhesion strength might affect cell shape, Barnhart et al. studied the effect of different adhesion strengths on the organization of adhesions, myosin II and the actin network in migrating keratocytes. On the basis of their results, they put forward a model in which cell shape and migratory behavior depend on regulation of the actin network — in particular, retrograde flow and polymerization — by adhesion and myosin, such that quantitative changes in either parameter evoke qualitative changes in migratory phenotype. The authors acknowledge that their mechanical model for cell shape determination does not include aspects of integrin signaling, but envisage that specific contributions of certain biochemical signaling pathways could be incorporated into future models.

• Barnhart EL, Lee K-C, Keren K, Mogilner A, Theriot JA. An adhesion-dependent switch between mechanisms that determine motile cell shape. PLoS Biol. 2011; 9(5): e1001059. PubMed

Pacemaker defines leading edge dynamics

The leading edge of migrating cells undergoes cycles of protrusion and retraction mediated by cytoskeletal remodeling, but the signaling components and pathways that regulate the timing of this cycling have not yet been elucidated. Tkachenko et al. now report on the identification of a three-component pacemaker — comprising protein kinase A (PKA), RhoA and Rho guanine dissociation inhibitor (RhoGDI) — that regulates the morphodynamic events at the leading edge of migrating cells. By using a pseudosubstrate peptide inhibitor, the authors found that PKA activity determines the magnitude, duration and transverse propagation of protrusions; furthermore, a membrane-bound biosensor indicated that PKA activity correlates temporally with membrane protrusion. RhoA is a PKA substrate, the activity of which at the leading edge also correlates with cell protrusion, and Tkachenko et al. found that PKA-mediated phosphorylation of RhoA on Ser188 regulated the dynamics of RhoA activation at the leading edge. Phosphorylation of RhoA is also known to increase its affinity for RhoGDI, thereby decreasing RhoA activity and, as overexpression of RhoA or a nonphosphorylatable form of RhoA (Ser188Ala) resulted in morphodynamics that resembled those seen following PKA inhibition, the authors surmised that the Rho–RhoGDI interaction mediated termination of protrusion. Finally, Tkachenko et al. established that adhesion was required for PKA activation during protrusion. Thus, under the elevated adhesion forces that balance the propulsive forces produced by actin filament assembly at the protruding cell edge, PKA phosphorylates Ser188 on RhoA. This increases the interaction between RhoA and RhoGDI, which displaces RhoA from the leading edge and abrogates effector interactions. The consequent loss of RhoA activity at the leading edge mediates the termination of protrusions, thereby defining the timing of a protrusion–retraction cycle.

PKA, RhoA, and RhoGDI form a protrusion–retraction pacemaker at the leading edge of migrating cells.

RhoA activity enables initiation of protrusion of the adherent leading edge, resulting in PKA activation. PKA phosphorylates RhoA at Ser188, leading to increased affinity for RhoGDI. RhoGDI binding both removes RhoA from the membrane and interferes with its coupling to effectors, which results in termination of RhoA activity and shortening duration of protrusions, thereby completing a self-inhibiting negative-feedback loop that acts as a pacemaker of the protrusion–retraction cycle. When PKA is inhibited or RhoA overexpression exceeds levels of available RhoGDI, protrusion duration and amplitude are markedly increased. This much slower cycle suggests the existence of a second slower default pacemaker, such as the RhoA GTP–GDP cycle, depicted in light gray.

• Tkachenko E, Sabouri-Ghomi M, Pertz O, Kim C, Gutierrez E, Machacek M, Groisman A, Danuser G, Ginsberg MH. Protein Kinase A Governs a RhoA-RhoGDI Protrusion-Retraction Pacemaker in Migrating Cells. Nat Cell Biol. 2011; [Epub] PubMed

RhoC concentrates cofilin at the core

Cancer cells are renowned for their ability to degrade and move through surrounding extracellular matrices and basement membranes, largely mediated by their ability to form F-actin-rich, matrix-degrading invadopodia. Actin dynamics must be regulated within these protrusions — in particular, cofilin is required to generate free barbed ends. Bravo-Cordero et al. now report in Current Biology how the spatial regulation of RhoC restricts the actin-severing activity of cofilin to within the invadopodium core to facilitate protrusion. Using a new biosensor for RhoC, the authors observed high RhoC activity in MTLn3 rat mammary adenocarcinoma cells in the area surrounding a forming invadopodium, rather than inside it. Conversely, the GTPase-activating protein p190RhoGAP localized within the invadopodium core to inhibit RhoC activity in this area, whereas the distribution of the guanine nucleotide-exchange factor p190RhoGEF in areas surrounding invadopodia mirrored the localization of active RhoC. The authors also observed that cofilin phosphorylated on Ser3 (pCofilinS3) — which is inactive — was enriched around, rather than inside, invadopodia, in wild-type cells, whereas it showed distribution throughout invadopodia in cells lacking RhoC. Furthermore, inhibiting either the activity of Rho kinase (ROCK) or the expression of LIM kinase (LIMK), which function downstream of RhoC, resulted in a decrease in the levels of pCofilinS3 and an increase in the number of barbed ends which were no longer focused in the core. So, by localizing RhoC activity to the area surrounding an invadopodium through the spatial regulation of p190RhoGAP and p190RhoGEF, cofilin phosphorylation by ROCK and LIMK can be similarly restricted. This leaves unphosphorylated, active cofilin to generate free barbed ends for actin remodeling within the invadopodium core, ultimately facilitating tumor cell invasion.

• Bravo-Cordero JJ, Oser M, Chen X, Eddy R, Hodgson L, Condeelis J. A novel spatiotemporal RhoC activation pathway locally regulates cofilin activity at invadopodia. Curr Biol. 2011; 21(8): 1–10 PubMed

ERK emerges as key coordinator

For cells to move forward, nascent adhesions must assemble and disassemble while lamellipodia protrude, but knowledge of how coordinated regulation of the cytoskeleton and adhesion machinery is achieved remains limited. Extracellular signal-regulated kinase (ERK) is known to regulate the disassembly of adhesions and is required for membrane protrusion, which prompted Mendoza et al. to investigate a role for ERK in linking protrusion with adhesion turnover. The authors first confirmed the requirement for ERK activity in protrusion and for components of the WAVE2 regulatory complex (WRC), which activates the Arp2/3 actin nucleator, in mediating this response. Although active ERK and WAVE2 colocalized at protruding edges, Mendoza et al. found that ERK does not regulate the activity of WRC by promoting its localization at the membrane; instead, ERK phosphorylates two components of the WRC - WAVE2 and Abi1 - that promote its interaction with Arp2/3 and actin. Mass spectrometry analysis identified several ERK phosphorylation sites within the proline-rich domains of WAVE2 and Abi1, which, when mutated to phosphomimetic residues, increased WRC coimmunoprecipitation with Arp3 and actin. Conversely, mutating the ERK phosphorylation sites on Abi1 to nonphosphorylatable alanines abrogated the WRC-Arp2/3 interaction and consequently inhibited protrusion and reduced migration speed in Cos-7 cells. Mendoza et al. propose that phosphorylation of WAVE2 and Abi1 by ERK might mediate lamellipodial protrusion by promoting the Arp2/3-mediated generation of an actin meshwork to provide a pushing force, and/or controlling the actin polymerization involved in nascent adhesion formation. Thus, signaling through ERK seems to coordinate lamellipodial protrusion-retraction with adhesion turnover.

• Mendoza MC, Er EE, Zhang W, Ballif BA, Elliott HL, Danuser G, Blenis J. ERK-MAPK drives lamellipodia protrusion by activating the WAVE2 regulatory complex. Mol. Cell 2011; PubMed

Integrin activation: regulation by extracellular pH

Changes in both the extracellular or intracellular environments can affect integrin activation. Acidic extracellular pH is known to influence cell adhesion and migration, but the molecular mechanisms responsible have not been elucidated, prompting Paradise et al. to investigate the potential effect of acidic extracellular pH on the activity of αvβ3 integrin. First, the researchers used the extracellular domain of αvβ3 to carry out molecular dynamics simulations, the results of which indicated that the αvβ3 headpiece opened more frequently (indicative of a high-affinity conformation) at acidic extracellular pH than at normal physiological pH (7.4). In the context of live cells, flow cytometry experiments using an antibody that preferentially binds to activated αvβ3 revealed an increase in the level of these receptors on the cell surface in response to exposure to an acidic pH. Atomic force microscope-mediated force spectroscopy studies also showed that the frequency of αvβ3–RGD interactions on live αvβ3-expressing CHO cells increased as a function of decreasing extracellular pH; the force required to rupture the ligand–receptor complex was unaffected by increased extracellular acidity. Consistent with increased extracellular acidity causing an increase in integrin activation, Paradise et al. found that lowering the pH of cell-culture media promoted cell adhesion and spreading, and that the concentration of fibronectin needed for cells to migrate efficiently was lower at acidic pH. As the authors highlight, the ability of the pH of the extracellular milieu to influence integrin activation and binding affinity is likely to affect the behavior of cells within regions of altered pH, such as within tumors or in wounded tissues.

• Paradise RK, Lauffenburger DA, Van Vliet KJ. Acidic extracellular pH promotes activation of integrin αvβ3. PLoS One 2011; 6 (1):e15746. PubMed

A selective sensor for reporting p38α activity in lysates

The Ser/Thr kinase p38α is implicated in several inflammatory diseases and in non-small cell lung carcinoma, but current methods to assess the activity of this mitogen-activated protein kinase family member often lack isoform specificity and require fractionated cell lysates. However, Barbara Imperiali and colleagues have now succeeded in designing and validating a p38α-specific activity probe for use in unfractionated cell lysates. The group had previously developed CSox, a sulfonamido-oxine fluorophore that increases in fluorescence in response to phosphorylation when placed (–)2 or (+)2 relative to the phosphorylation site in an optimized kinase substrate, owing to chelation of a magnesium ion between it and the newly added phosphoryl group. In their current report, in ACS Chemical Biology, they linked a known docking peptide sequence to a CSox-based phosphorylation site via a flexible linker. In vitro, the sensor — MEF2A-CSox — was selectively phosphorylated by p38α, displaying minimal background activity in the presence of several other related kinases. Significantly, MEF2A-CSox was able to report p38α activation in the unfractionated lysates of HeLa cells in response to tumor necrosis factor (an inflammatory agent) or sorbitol (to induce osmotic shock). Addition of SB203580 or BIRB796, both of which inhibit p38α, led to a decrease in MEF2A-CSox fluorescence in a dose-dependent manner, and background subtraction of parallel reactions to which 1 μM SB203580 was added could be used to eliminate background signal. As Imperiali and colleagues showed that MEF2A-CSox could report the activation of p38α in several cell lines taken from different species, this isoform-selective sensor seems to have widespread potential use in both research and clinical settings.

• Stains CI, Luković E, Imperiali B. A p38α-selective chemosensor for use in unfractionated cell lysates. ACS Chem Biol. 2010;6(1):101-105. PubMed

Stretching to the myosin rhythm

During the development of many organs, dramatic changes in tissue architecture occur — for example, small round buds elongate to form highly elaborate structures during mammary gland development. The mechanisms that regulate such changes in overall organ shape are not well understood. Such tissue elongation also occurs in the Drosophila ovary, where — between developmental stages 8 and 10 — egg chambers elongate substantially while also increasing in volume. By combining live imaging and egg-chamber culturing techniques, Denise Montell’s group now report that elongation of the egg chamber requires directional, oscillating contractions of the actomyosin network near the basal surfaces of follicle cells. They showed that basal myosin accumulated and disappeared from individual cells, directly preceding changes in the basal cell surface area and the length of cells along the dorsal–ventral axis. Decreasing the levels of basal actin and myosin filaments blocked these myosin oscillations and resulted in wider egg chambers, whereas promoting actomyosin filament contraction increased basal myosin levels and restricted egg chamber width, thereby causing elongation. Myosin accumulation required Rho, Rho kinase and cytosolic calcium, whereas changes in cell–extracellular matrix adhesion affected the oscillation period as well as amplitude. So, whereas reducing talin expression decreased basal myosin levels, shortened the oscillation period and resulted in round egg chambers, these organs elongated in response to paxillin overexpression, consistent with increased myosin intensity and a longer oscillation period. The study overall reveals a mechanism whereby a dynamic ‘corset’, comprising the periodic assembly and disassembly of myosin on actin filaments, can control organ shape. In this case, contraction of the corset generates a force that propagates inwards, opposing the growth-mediated outward force, and expansion is directed to the poles of the egg chamber.

Other reviews of this article are available here and here.

• He L, Wang X, Tang HL, Montell DJ. Tissue elongation requires oscillating contractions of a basal actomyosin network. Nat. Cell Biol. 2010; 12 (12); 1133–42. PubMed