Reconstitution of amoeboid motility in vitro identifies a motor-independent mechanism for cell body retraction

Crawling movement in eukaryotic cells requires coordination of leading-edge protrusion with cell body retraction [1-3]. Protrusion is driven by actin polymerization along the leading edge [4]. The mechanism of retraction is less clear; myosin contractility may be involved in some cells [5] but is not essential in others [6-9]. In Ascaris sperm, protrusion and retraction are powered by the major sperm protein (MSP) motility system instead of the conventional actin apparatus [10, 11]. These cells lack motor proteins [12] and so are well suited to explore motor-independent mechanisms of retraction. We reconstituted protrusion and retraction simultaneously in MSP filament meshworks, called fibers, that assemble behind plasma membrane-derived vesicles. Retraction is triggered by depolymerization of complete filaments in the rear of the fiber [13]. The surviving filaments reorganize to maintain their packing density. By packing fewer filaments into a smaller volume, the depolymerizing network shrinks and thereby generates sufficient force to move an attached load. Our work provides direct evidence for motor-independent retraction in the reconstituted MSP motility system of nematode sperm. This mechanism could also apply to actin-based cells and may explain reports of cells that crawl even when their myosin activity is compromised.     

Shaping fission yeast cells by rerouting actin-based transport on microtubules

Kinesins and myosins transport cargos to specific locations along microtubules and actin filaments, respectively. The relative contribution of the two transport systems for cell polarization varies extensively in different cell types, with some cells relying exclusively on actin-based transport while others mainly use microtubules. Using fission yeast, we asked whether one transport system can substitute for the other. In this organism, microtubules and actin cables both contribute to polarized growth by transporting cargos to cell poles, but with distinct roles: microtubules transport landmarks to label cell poles for growth and actin assembly but do not directly contribute to the growth process [1]. Actin cables serve as tracks for myosin V delivery of growth vesicles to cell poles [ [2] , [3] and [4] ]. We engineered a chimera between the motor domain of the kinesin 7 Tea2 and the globular tail of the myosin V Myo52, which we show transports Ypt3, a myosin cargo receptor, to cell poles along microtubules. Remarkably, this chimera restores polarized growth and viability to cells lacking actin cables. It also bypasses the normal microtubule-dependent marking of cell poles for polarized growth, but not for other functions. Thus, a synthetic motor protein successfully redirects cargos along a distinct cytoskeletal route.

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3.

Tor complex 1 controls telomere length by affecting the level of Ku     

Ungar L  Harari Y  Toren A  Kupiec M 《Current biology : CB》2011,21(24):2115-2120

Telomeres are specialized DNA-protein structures at the ends of eukaryotic chromosomes. Telomeric DNA is synthesized by telomerase, which is expressed only at the early stages of development [ [1] and [2] ]. To become malignant, any cell has to be able to replenish telomeres [3]. Thus, understanding how telomere length is monitored has significant medical implications, especially in the fields of aging and cancer. In yeast, telomerase is constitutively active. A large network of genes participates in controlling telomere length [ [4] , [5] , [6] , [7] and [8] ]. Tor1 and Tor2 (targets of rapamycin [9]) are two similar kinases that regulate cell growth [10]. Both can be found as part of the TOR complex 1 (TORC1 [11]), which coordinates the response to nutrient starvation and is sensitive to rapamycin [12]. The rapamycin-insensitive TOR complex 2 (TORC2) contains only Tor2 and regulates actin cytoskeleton polarization [13]. Here we provide evidence for a role of TORC1 in telomere shortening upon starvation in yeast cells. The TORC1 signal is transduced by the Gln3/Gat1/Ure2 pathway, which controls the levels of the Ku heterodimer, a telomere regulator. We discuss the potential implications for the usage of rapamycin as a therapeutic agent against cancer and the effect that calorie restriction may have on telomere length.     

4.

Cortactin releases the brakes in actin- based motility by enhancing WASP-VCA detachment from Arp2/3 branches     

Siton O  Ideses Y  Albeck S  Unger T  Bershadsky AD  Gov NS  Bernheim-Groswasser A 《Current biology : CB》2011,21(24):2092-2097

Cortactin is involved in invadopodia and podosome formation [1], pathogens and endosome motility [2], and persistent lamellipodia protrusion [ [3] and [4] ]; its overexpression enhances cellular motility and metastatic activity [ [5] , [6] , [7] and [8] ]. Several mechanisms have been proposed to explain cortactin's role in Arp2/3-driven actin polymerization [ [9] and [10] ], yet its direct role in cell movement remains unclear. We use a biomimetic system to study the mechanism of cortactin-mediated regulation of actin-driven motility [11]. We tested the role of different cortactin variants that interact with Arp2/3 complex and actin filaments distinctively. We show that wild-type cortactin significantly enhances the bead velocity at low concentrations. Single filament experiments show that cortactin has no significant effect on actin polymerization and branch stability, whereas it strongly affects the branching rate driven by Wiskott-Aldrich syndrome protein (WASP)-VCA fragment and Arp2/3 complex. These results lead us to propose that cortactin plays a critical role in translating actin polymerization at a bead surface into motion, by releasing WASP-VCA from the new branching site. This enhanced release has two major effects: it increases the turnover rate of branching per WASP molecule, and it decreases the friction-like force caused by the binding of the moving surface with respect to the growing actin network.     

5.

Enforced polarisation and locomotion of fibroblasts lacking microtubules     

Kaverina I  Krylyshkina O  Gimona M  Beningo K  Wang YL  Small JV 《Current biology : CB》2000,10(12):739-742

The polarisation and locomotion of fibroblasts requires an intact microtubule cytoskeleton [1]. This has been attributed to an influence of microtubule-mediated signals on actin cytoskeleton dynamics, either through the generation of active Rac to promote protrusion of lamellipodia [2], or through the modulation of substrate adhesion via microtubule targeting events [3] [4]. We show here that the polarizing role of microtubules can be mimicked by externally imposing an asymmetric gradient of contractility by local application of the contractility inhibitor ML-7. Apolar fibroblasts lacking microtubules could be induced to polarize and to move by application of ML-7 by micropipette to one side of the cell and then to the trailing vertices that developed. The release and retraction of trailing adhesions could be correlated with a relaxation of traction on the substrate and a differential shortening of stress-fibre bundles, with their distal tips relaxed. Although retraction and protrusion in these conditions resembled control cell locomotion, the normal turnover of adhesion sites that form behind the protruding cell front was blocked. These findings show that microtubules are dispensable for fibroblast protrusion, but are required for the turnover of substrate adhesions that normally occurs during cell locomotion. We conclude that regional contractility is modulated by the interfacing of microtubule-linked events with focal adhesions and that microtubules determine cell polarity via this route.     

6.

Myosin II redistribution during rear retraction and the role of filament assembly and disassembly     

Koehl G  McNally JG 《Cell biology international》2002,26(3):287-296

The literature to date suggests a role for myosin II in rear retraction, including evidence that myosin undergoes a characteristic 'C'-to-spot redistribution at the cell posterior which is associated with retraction. Here we investigate the mechanism of both retraction and the'C'-to-spot using Dictyostelium cells containing mutant forms of myosin that affect its polymerization. 3 x Asp-myosin forms few if any filaments. When 3 x Asp cells are added to a wild-type mound, the mutant cells move directionally, but rear retraction is markedly delayed,demonstrating that myosin II filaments are essential for efficient retraction. In addition, using a GFP-tagged 3 x Asp-myosin, we observed a posterior spot pattern associated with retraction,but no cortical 'C' pattern preceding it. This suggests that filamentous myosin is required to produce the 'C', and that its failure to form results in defective rear retraction. In contrast, an alternate mutant myosin that forms filaments constitutively, 3 x Ala-myosin, forms 'Cs' and then spot patterns at the posterior, but in the interim the spots do not disintegrate. This suggests that spot dissolution occurs by filament depolymerization. In summary our data demonstrate a role for myosin II and the 'C'-to-spot in efficient rear retraction, and define filament assembly as critical for formation of the 'C' and filament disassembly as critical for dissolution of the spot.     

7.

Myosin IIA regulates cell motility and actomyosin-microtubule crosstalk     

Even-Ram S  Doyle AD  Conti MA  Matsumoto K  Adelstein RS  Yamada KM 《Nature cell biology》2007,9(3):299-309

Non-muscle myosin II has diverse functions in cell contractility, cytokinesis and locomotion, but the specific contributions of its different isoforms have yet to be clarified. Here, we report that ablation of the myosin IIA isoform results in pronounced defects in cellular contractility, focal adhesions, actin stress fibre organization and tail retraction. Nevertheless, myosin IIA-deficient cells display substantially increased cell migration and exaggerated membrane ruffling, which was dependent on the small G-protein Rac1, its activator Tiam1 and the microtubule moter kinesin Eg5. Myosin IIA deficiency stabilized microtubules, shifting the balance between actomyosin and microtubules with increased microtubules in active membrane ruffles. When microtubule polymerization was suppressed, myosin IIB could partially compensate for the absence of the IIA isoform in cellular contractility, but not in cell migration. We conclude that myosin IIA negatively regulates cell migration and suggest that it maintains a balance between the actomyosin and microtubule systems by regulating microtubule dynamics.     

8.

The role of actin turnover in retrograde actin network flow in neuronal growth cones     

Van Goor D  Hyland C  Schaefer AW  Forscher P 《PloS one》2012,7(2):e30959

The balance of actin filament polymerization and depolymerization maintains a steady state network treadmill in neuronal growth cones essential for motility and guidance. Here we have investigated the connection between depolymerization and treadmilling dynamics. We show that polymerization-competent barbed ends are concentrated at the leading edge and depolymerization is distributed throughout the peripheral domain. We found a high-to-low G-actin gradient between peripheral and central domains. Inhibiting turnover with jasplakinolide collapsed this gradient and lowered leading edge barbed end density. Ultrastructural analysis showed dramatic reduction of leading edge actin filament density and filament accumulation in central regions. Live cell imaging revealed that the leading edge retracted even as retrograde actin flow rate decreased exponentially. Inhibition of myosin II activity before jasplakinolide treatment lowered baseline retrograde flow rates and prevented leading edge retraction. Myosin II activity preferentially affected filopodial bundle disassembly distinct from the global effects of jasplakinolide on network turnover. We propose that growth cone retraction following turnover inhibition resulted from the persistence of myosin II contractility even as leading edge assembly rates decreased. The buildup of actin filaments in central regions combined with monomer depletion and reduced polymerization from barbed ends suggests a mechanism for the observed exponential decay in actin retrograde flow. Our results show that growth cone motility is critically dependent on continuous disassembly of the peripheral actin network.     

9.

Transcriptomes of the parasitic plant family Orobanchaceae reveal surprising conservation of chlorophyll synthesis     

Wickett NJ  Honaas LA  Wafula EK  Das M  Huang K  Wu B  Landherr L  Timko MP  Yoder J  Westwood JH  dePamphilis CW 《Current biology : CB》2011,21(24):2098-2104

     

10.

An adhesion-dependent switch between mechanisms that determine motile cell shape     

Barnhart EL  Lee KC  Keren K  Mogilner A  Theriot JA 《PLoS biology》2011,9(5):e1001059

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.     

11.

Actin stress fibre subtypes in mesenchymal-migrating cells     

Tea Vallenius 《Open biology》2013,3(6)

Mesenchymal cell migration is important for embryogenesis and tissue regeneration. In addition, it has been implicated in pathological conditions such as the dissemination of cancer cells. A characteristic of mesenchymal-migrating cells is the presence of actin stress fibres, which are thought to mediate myosin II-based contractility in close cooperation with associated focal adhesions. Myosin II-based contractility regulates various cellular activities, which occur in a spatial and temporal manner to achieve directional cell migration. These myosin II-based activities involve the maturation of integrin-based adhesions, generation of traction forces, establishment of the front-to-back polarity axis, retraction of the trailing edge, extracellular matrix remodelling and mechanotransduction. Growing evidence suggests that actin stress fibre subtypes, namely dorsal stress fibres, transverse arcs and ventral stress fibres, could provide this spatial and temporal myosin II-based activity. Consistent with their functional differences, recent studies have demonstrated that the molecular composition of actin stress fibre subtypes differ significantly. This present review focuses on the current view of the molecular composition of actin stress fibre subtypes and how these fibre subtypes regulate mesenchymal cell migration.     

12.

Optical reversal of halothane-induced immobility in C. elegans     

Singaram VK  Somerlot BH  Falk SA  Falk MJ  Sedensky MM  Morgan PG 《Current biology : CB》2011,21(24):2070-2076

Volatile anesthetics (VAs) cause profound neurological effects, including reversible loss of consciousness and immobility. Despite their widespread use, the mechanism of action of VAs remains one of the unsolved puzzles of neuroscience [ [1] and [2] ]. Genetic studies in Caenorhabditis elegans [ [3] and [4] ], Drosophila [ [3] and [5] ], and mice [ [6] , [7] , [8] and [9] ] indicate that ion channels controlling the neuronal resting membrane potential (RMP) also control anesthetic sensitivity. Leak channels selective for K⁺ [ [10] , [11] , [12] and [13] ] or permeable to Na⁺ [14] are critical for establishing RMP. We hypothesized that halothane, a VA, caused immobility by altering the neuronal RMP. In C. elegans, halothane-induced immobility is acutely and completely reversed by channelrhodopsin-2 based depolarization of the RMP when expressed specifically in cholinergic neurons. Furthermore, hyperpolarizing cholinergic neurons via halorhodopsin activation increases sensitivity to halothane. The sensitivity of C. elegans to halothane can be altered by 25-fold by either manipulation of membrane conductance with optogenetic methods or generation of mutations in leak channels that set the RMP. Immobility induced by another VA, isoflurane, is not affected by these treatments, thereby excluding the possibility of nonspecific hyperactivity. The sum of our data indicates that leak channels and the RMP are important determinants of halothane-induced general anesthesia.     

13.

Asymmetric distribution of myosin IIB in migrating endothelial cells is regulated by a rho-dependent kinase and contributes to tail retraction          下载免费PDF全文

Kolega J 《Molecular biology of the cell》2003,14(12):4745-4757

All vertebrates contain two nonmuscle myosin II heavy chains, A and B, which differ in tissue expression and subcellular distributions. To understand how these distinct distributions are controlled and what role they play in cell migration, myosin IIA and IIB were examined during wound healing by bovine aortic endothelial cells. Immunofluorescence showed that myosin IIA skewed toward the front of migrating cells, coincident with actin assembly at the leading edge, whereas myosin IIB accumulated in the rear 15-30 min later. Inhibition of myosin light-chain kinase, protein kinases A, C, and G, tyrosine kinase, MAP kinase, and PIP3 kinase did not affect this asymmetric redistribution of myosin isoforms. However, posterior accumulation of myosin IIB, but not anterior distribution of myosin IIA, was inhibited by dominant-negative rhoA and by the rho-kinase inhibitor, Y-27632, which also inhibited myosin light-chain phosphorylation. This inhibition was overcome by transfecting cells with constitutively active myosin light-chain kinase. These observations indicate that asymmetry of myosin IIB, but not IIA, is regulated by light-chain phosphorylation mediated by rho-dependent kinase. Blocking this pathway inhibited tail constriction and retraction, but did not affect protrusion, suggesting that myosin IIB functions in pulling the rear of the cell forward.     

14.

A selective difference between human Y-chromosomal DNA haplotypes     

M.A. Jobling  G. Williams  K. Schiebel  A. Pandya  K. McElreavey  L. Salas  G.A. Rappold  N.A. Affara  C. Tyler-SmithAuthor vitae 《Current biology : CB》1998,8(25):1391

DNA analysis is making a valuable contribution to the understanding of human evolution [1]. Much attention has focused on mitochondrial DNA (mtDNA) [2] and the Y chromosome [3] and [4], both of which escape recombination and so provide information on maternal and paternal lineages, respectively. It is often assumed that the polymorphisms observed at loci on mtDNA and the Y chromosome are selectively neutral and, therefore, that existing patterns of molecular variation can be used to deduce the histories of populations in terms of drift, population movements, and cultural practices. The coalescence of the molecular phylogenies of mtDNA and the Y chromosome to recent common ancestors in Africa [5] and [6], for example, has been taken to reflect a recent origin of modern human populations in Africa. An alternative explanation, though, could be the recent selective spread of mtDNA and Y chromosome haplotypes from Africa in a population with a more complex history [7]. It is therefore important to establish whether there are selective differences between classes (haplotypes) of mtDNA and Y chromosomes and, if so, whether these differences could have been sufficient to influence the distributions of haplotypes in existing populations. A precedent for this hypothesis has been established for mtDNA in that one mtDNA background increases susceptibility to Leber hereditary optic neuropathy [8]. Although studies of nucleotide diversity in global samples of Y chromosomes have suggested an absence of recent selective sweeps or bottlenecks [9], selection may, in principle, be very important for the Y chromosome because it carries several loci affecting male fertility [10] and [11] and as many as 5% of males are infertile [11] and [12]. Here, we show that one class of infertile males, PRKX/PRKY translocation XX males, arises predominantly on a particular Y haplotypic background. Selection is, therefore, acting on Y haplotype distributions in the population.     

15.

Recruitment of a myosin heavy chain kinase to actin-rich protrusions in Dictyostelium     

Steimle PA  Yumura S  Côté GP  Medley QG  Polyakov MV  Leppert B  Egelhoff TT 《Current biology : CB》2001,11(9):708-713

Nonmuscle myosin II plays fundamental roles in cell body translocation during migration and is typically depleted or absent from actin-based cell protrusions such as lamellipodia, but the mechanisms preventing myosin II assembly in such structures have not been identified [1-3]. In Dictyostelium discoideum, myosin II filament assembly is controlled primarily through myosin heavy chain (MHC) phosphorylation. The phosphorylation of sites in the myosin tail domain by myosin heavy chain kinase A (MHCK A) drives the disassembly of myosin II filaments in vitro and in vivo [4]. To better understand the cellular regulation of MHCK A activity, and thus the regulation of myosin II filament assembly, we studied the in vivo localization of native and green fluorescent protein (GFP)-tagged MHCK A. MHCK A redistributes from the cytosol to the cell cortex in response to stimulation of Dictyostelium cells with chemoattractant in an F-actin-dependent manner. During chemotaxis, random migration, and phagocytic/endocytic events, MHCK A is recruited preferentially to actin-rich leading-edge extensions. Given the ability of MHCK A to disassemble myosin II filaments, this localization may represent a fundamental mechanism for disassembling myosin II filaments and preventing localized filament assembly at sites of actin-based protrusion.     

16.

p116Rip targets myosin phosphatase to the actin cytoskeleton and is essential for RhoA/ROCK-regulated neuritogenesis          下载免费PDF全文

Mulder J  Ariaens A  van den Boomen D  Moolenaar WH 《Molecular biology of the cell》2004,15(12):5516-5527

Activation of the RhoA-Rho kinase (ROCK) pathway stimulates actomyosin-driven contractility in many cell systems, largely through ROCK-mediated inhibition of myosin II light chain phosphatase. In neuronal cells, the RhoA-ROCK-actomyosin pathway signals cell rounding, growth cone collapse, and neurite retraction; conversely, inhibition of RhoA/ROCK promotes cell spreading and neurite outgrowth. The actin-binding protein p116(Rip), whose N-terminal region bundles F-actin in vitro, has been implicated in Rho-dependent neurite remodeling; however, its function is largely unknown. Here, we show that p116(Rip), through its C-terminal coiled-coil domain, interacts directly with the C-terminal leucine zipper of the regulatory myosin-binding subunits of myosin II phosphatase, MBS85 and MBS130. RNA interference-induced knockdown of p116(Rip) inhibits cell spreading and neurite outgrowth in response to extracellular cues, without interfering with the regulation of myosin light chain phosphorylation. We conclude that p116(Rip) is essential for neurite outgrowth and may act as a scaffold to target the myosin phosphatase complex to the actin cytoskeleton.     

17.

Cellular Regulation of Extension and Retraction of Pseudopod-Like Blebs Produced by Nanosecond Pulsed Electric Field (nsPEF)     

Mikhail A. Rassokhin  Andrei G. Pakhomov 《Cell biochemistry and biophysics》2014,69(3):555-566

Recently we described a new phenomenon of anodotropic pseudopod-like blebbing in U937 cells exposed to nanosecond pulsed electric field (nsPEF). In Ca²⁺-free buffer such exposure initiates formation of pseudopod-like blebs (PLBs), protrusive cylindrical cell extensions that are distinct from apoptotic and necrotic blebs. PLBs nucleate predominantly on anode-facing cell pole and extend toward anode during nsPEF exposure. Bleb extension depends on actin polymerization and availability of actin monomers. Inhibition of intracellular Ca²⁺, cell contractility, and RhoA produced no effect on PLB initiation. Meanwhile, inhibition of WASP by wiskostatin causes dose-dependent suppression of PLB growth. Soon after the end of nsPEF exposure PLBs lose directionality of growth and then retract. Microtubule toxins nocodazole and paclitaxel did not show immediate effect on PLBs; however, nocodazole increased mobility of intracellular components during PLB extension and retraction. Retraction of PLBs is produced by myosin activation and the corresponding increase in PLB cortex contractility. Inhibition of myosin by blebbistatin reduces retraction while inhibition of RhoA–ROCK pathway by Y-27632 completely prevents retraction. Contraction of PLBs can produce cell translocation resembling active cell movement. Overall, the formation, properties, and life cycle of PLBs share common features with protrusions associated with ameboid cell migration. PLB life cycle may be controlled through activation of WASP by its upstream effectors such as Cdc42 and PIP2, and main ROCK activator—RhoA. Parallels between pseudopod-like blebbing and motility blebbing may provide new insights into their underlying mechanisms.     

18.

A cytoskeletal demolition worker: myosin II acts as an actin depolymerization agent     

Haviv L  Gillo D  Backouche F  Bernheim-Groswasser A 《Journal of molecular biology》2008,375(2):325-330

Myosin II motors play several important roles in a variety of cellular processes, some of which involve active assembly/disassembly of cytoskeletal substructures. Myosin II motors have been shown to function in actin bundle turnover in neuronal growth cones and in the recycling of actin filaments during cytokinesis. Close examination had shown an intimate relationship between myosin II motor adenosine triphosphatase activity and actin turnover rate. However, the direct implication of myosin II in actin turnover is still not understood. Herein, we show, using high-resolution cryo-transmission electron microscopy, that myosin II motors control the turnover of actin bundles in a concentration-dependent manner in vitro. We demonstrate that disassembly of actin bundles occurs through two main stages: the first stage involves unbundling into individual filaments, and the second involves their subsequent depolymerization. These evidence suggest that, in addition to their “classical” contractile abilities, myosin II motors may be directly implicated in active actin depolymerization. We believe that myosin II motors may function similarly in vivo (e.g., in the disassembly of the contractile ring by fine tuning the local concentration/activity of myosin II motors).     

19.

The RhoGEF TEM4 Regulates Endothelial Cell Migration by Suppressing Actomyosin Contractility     

Natalia Mitin  Kent L. Rossman  Rachel Currin  Sandeep Anne  Thomas W. Marshall  James E. Bear  Victoria L. Bautch  Channing J. Der 《PloS one》2013,8(6)

Persistent cellular migration requires efficient protrusion of the front of the cell, the leading edge where the actin cytoskeleton and cell-substrate adhesions undergo constant rearrangement. Rho family GTPases are essential regulators of the actin cytoskeleton and cell adhesion dynamics. Here, we examined the role of the RhoGEF TEM4, an activator of Rho family GTPases, in regulating cellular migration of endothelial cells. We found that TEM4 promotes the persistence of cellular migration by regulating the architecture of actin stress fibers and cell-substrate adhesions in protruding membranes. Furthermore, we determined that TEM4 regulates cellular migration by signaling to RhoC as suppression of RhoC expression recapitulated the loss-of-TEM4 phenotypes, and RhoC activation was impaired in TEM4-depleted cells. Finally, we showed that TEM4 and RhoC antagonize myosin II-dependent cellular contractility and the suppression of myosin II activity rescued the persistence of cellular migration of TEM4-depleted cells. Our data implicate TEM4 as an essential regulator of the actin cytoskeleton that ensures proper membrane protrusion at the leading edge of migrating cells and efficient cellular migration via suppression of actomyosin contractility.     

20.

Acquiring "the Knowledge" of London's layout drives structural brain changes     

Woollett K  Maguire EA 《Current biology : CB》2011,21(24):2109-2114

The last decade has seen a burgeoning of reports associating brain structure with specific skills and traits (e.g., [ [1] , [2] , [3] , [4] , [5] , [6] , [7] and [8] ]). Although these cross-sectional studies are informative, cause and effect are impossible to establish without longitudinal investigation of the same individuals before and after an intervention. Several longitudinal studies have been conducted (e.g., [ [9] , [10] , [11] , [12] , [13] , [14] , [15] , [16] , [17] and [18] ]); some involved children or young adults, potentially conflating brain development with learning, most were restricted to the motor domain, and all concerned relatively short timescales (weeks or months). Here, by contrast, we utilized a unique opportunity to study average-IQ adults operating in the real world as they learned, over four years, the complex layout of London's streets while training to become licensed taxi drivers. In those who qualified, acquisition of an internal spatial representation of London was associated with a selective increase in gray matter (GM) volume in their posterior hippocampi and concomitant changes to their memory profile. No structural brain changes were observed in trainees who failed to qualify or control participants. We conclude that specific, enduring, structural brain changes in adult humans can be induced by biologically relevant behaviors engaging higher cognitive functions such as spatial memory, with significance for the “nature versus nurture” debate.     

Tor complex 1 controls telomere length by affecting the level of Ku

Telomeres are specialized DNA-protein structures at the ends of eukaryotic chromosomes. Telomeric DNA is synthesized by telomerase, which is expressed only at the early stages of development [ [1] and [2] ]. To become malignant, any cell has to be able to replenish telomeres [3]. Thus, understanding how telomere length is monitored has significant medical implications, especially in the fields of aging and cancer. In yeast, telomerase is constitutively active. A large network of genes participates in controlling telomere length [ [4] , [5] , [6] , [7] and [8] ]. Tor1 and Tor2 (targets of rapamycin [9]) are two similar kinases that regulate cell growth [10]. Both can be found as part of the TOR complex 1 (TORC1 [11]), which coordinates the response to nutrient starvation and is sensitive to rapamycin [12]. The rapamycin-insensitive TOR complex 2 (TORC2) contains only Tor2 and regulates actin cytoskeleton polarization [13]. Here we provide evidence for a role of TORC1 in telomere shortening upon starvation in yeast cells. The TORC1 signal is transduced by the Gln3/Gat1/Ure2 pathway, which controls the levels of the Ku heterodimer, a telomere regulator. We discuss the potential implications for the usage of rapamycin as a therapeutic agent against cancer and the effect that calorie restriction may have on telomere length.     

Cortactin releases the brakes in actin- based motility by enhancing WASP-VCA detachment from Arp2/3 branches

Cortactin is involved in invadopodia and podosome formation [1], pathogens and endosome motility [2], and persistent lamellipodia protrusion [ [3] and [4] ]; its overexpression enhances cellular motility and metastatic activity [ [5] , [6] , [7] and [8] ]. Several mechanisms have been proposed to explain cortactin's role in Arp2/3-driven actin polymerization [ [9] and [10] ], yet its direct role in cell movement remains unclear. We use a biomimetic system to study the mechanism of cortactin-mediated regulation of actin-driven motility [11]. We tested the role of different cortactin variants that interact with Arp2/3 complex and actin filaments distinctively. We show that wild-type cortactin significantly enhances the bead velocity at low concentrations. Single filament experiments show that cortactin has no significant effect on actin polymerization and branch stability, whereas it strongly affects the branching rate driven by Wiskott-Aldrich syndrome protein (WASP)-VCA fragment and Arp2/3 complex. These results lead us to propose that cortactin plays a critical role in translating actin polymerization at a bead surface into motion, by releasing WASP-VCA from the new branching site. This enhanced release has two major effects: it increases the turnover rate of branching per WASP molecule, and it decreases the friction-like force caused by the binding of the moving surface with respect to the growing actin network.     

Enforced polarisation and locomotion of fibroblasts lacking microtubules

The polarisation and locomotion of fibroblasts requires an intact microtubule cytoskeleton [1]. This has been attributed to an influence of microtubule-mediated signals on actin cytoskeleton dynamics, either through the generation of active Rac to promote protrusion of lamellipodia [2], or through the modulation of substrate adhesion via microtubule targeting events [3] [4]. We show here that the polarizing role of microtubules can be mimicked by externally imposing an asymmetric gradient of contractility by local application of the contractility inhibitor ML-7. Apolar fibroblasts lacking microtubules could be induced to polarize and to move by application of ML-7 by micropipette to one side of the cell and then to the trailing vertices that developed. The release and retraction of trailing adhesions could be correlated with a relaxation of traction on the substrate and a differential shortening of stress-fibre bundles, with their distal tips relaxed. Although retraction and protrusion in these conditions resembled control cell locomotion, the normal turnover of adhesion sites that form behind the protruding cell front was blocked. These findings show that microtubules are dispensable for fibroblast protrusion, but are required for the turnover of substrate adhesions that normally occurs during cell locomotion. We conclude that regional contractility is modulated by the interfacing of microtubule-linked events with focal adhesions and that microtubules determine cell polarity via this route.     

Myosin II redistribution during rear retraction and the role of filament assembly and disassembly

The literature to date suggests a role for myosin II in rear retraction, including evidence that myosin undergoes a characteristic 'C'-to-spot redistribution at the cell posterior which is associated with retraction. Here we investigate the mechanism of both retraction and the'C'-to-spot using Dictyostelium cells containing mutant forms of myosin that affect its polymerization. 3 x Asp-myosin forms few if any filaments. When 3 x Asp cells are added to a wild-type mound, the mutant cells move directionally, but rear retraction is markedly delayed,demonstrating that myosin II filaments are essential for efficient retraction. In addition, using a GFP-tagged 3 x Asp-myosin, we observed a posterior spot pattern associated with retraction,but no cortical 'C' pattern preceding it. This suggests that filamentous myosin is required to produce the 'C', and that its failure to form results in defective rear retraction. In contrast, an alternate mutant myosin that forms filaments constitutively, 3 x Ala-myosin, forms 'Cs' and then spot patterns at the posterior, but in the interim the spots do not disintegrate. This suggests that spot dissolution occurs by filament depolymerization. In summary our data demonstrate a role for myosin II and the 'C'-to-spot in efficient rear retraction, and define filament assembly as critical for formation of the 'C' and filament disassembly as critical for dissolution of the spot.     

Myosin IIA regulates cell motility and actomyosin-microtubule crosstalk

Non-muscle myosin II has diverse functions in cell contractility, cytokinesis and locomotion, but the specific contributions of its different isoforms have yet to be clarified. Here, we report that ablation of the myosin IIA isoform results in pronounced defects in cellular contractility, focal adhesions, actin stress fibre organization and tail retraction. Nevertheless, myosin IIA-deficient cells display substantially increased cell migration and exaggerated membrane ruffling, which was dependent on the small G-protein Rac1, its activator Tiam1 and the microtubule moter kinesin Eg5. Myosin IIA deficiency stabilized microtubules, shifting the balance between actomyosin and microtubules with increased microtubules in active membrane ruffles. When microtubule polymerization was suppressed, myosin IIB could partially compensate for the absence of the IIA isoform in cellular contractility, but not in cell migration. We conclude that myosin IIA negatively regulates cell migration and suggest that it maintains a balance between the actomyosin and microtubule systems by regulating microtubule dynamics.     

The role of actin turnover in retrograde actin network flow in neuronal growth cones

The balance of actin filament polymerization and depolymerization maintains a steady state network treadmill in neuronal growth cones essential for motility and guidance. Here we have investigated the connection between depolymerization and treadmilling dynamics. We show that polymerization-competent barbed ends are concentrated at the leading edge and depolymerization is distributed throughout the peripheral domain. We found a high-to-low G-actin gradient between peripheral and central domains. Inhibiting turnover with jasplakinolide collapsed this gradient and lowered leading edge barbed end density. Ultrastructural analysis showed dramatic reduction of leading edge actin filament density and filament accumulation in central regions. Live cell imaging revealed that the leading edge retracted even as retrograde actin flow rate decreased exponentially. Inhibition of myosin II activity before jasplakinolide treatment lowered baseline retrograde flow rates and prevented leading edge retraction. Myosin II activity preferentially affected filopodial bundle disassembly distinct from the global effects of jasplakinolide on network turnover. We propose that growth cone retraction following turnover inhibition resulted from the persistence of myosin II contractility even as leading edge assembly rates decreased. The buildup of actin filaments in central regions combined with monomer depletion and reduced polymerization from barbed ends suggests a mechanism for the observed exponential decay in actin retrograde flow. Our results show that growth cone motility is critically dependent on continuous disassembly of the peripheral actin network.     

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.     

Actin stress fibre subtypes in mesenchymal-migrating cells

Mesenchymal cell migration is important for embryogenesis and tissue regeneration. In addition, it has been implicated in pathological conditions such as the dissemination of cancer cells. A characteristic of mesenchymal-migrating cells is the presence of actin stress fibres, which are thought to mediate myosin II-based contractility in close cooperation with associated focal adhesions. Myosin II-based contractility regulates various cellular activities, which occur in a spatial and temporal manner to achieve directional cell migration. These myosin II-based activities involve the maturation of integrin-based adhesions, generation of traction forces, establishment of the front-to-back polarity axis, retraction of the trailing edge, extracellular matrix remodelling and mechanotransduction. Growing evidence suggests that actin stress fibre subtypes, namely dorsal stress fibres, transverse arcs and ventral stress fibres, could provide this spatial and temporal myosin II-based activity. Consistent with their functional differences, recent studies have demonstrated that the molecular composition of actin stress fibre subtypes differ significantly. This present review focuses on the current view of the molecular composition of actin stress fibre subtypes and how these fibre subtypes regulate mesenchymal cell migration.     

Optical reversal of halothane-induced immobility in C. elegans

Volatile anesthetics (VAs) cause profound neurological effects, including reversible loss of consciousness and immobility. Despite their widespread use, the mechanism of action of VAs remains one of the unsolved puzzles of neuroscience [ [1] and [2] ]. Genetic studies in Caenorhabditis elegans [ [3] and [4] ], Drosophila [ [3] and [5] ], and mice [ [6] , [7] , [8] and [9] ] indicate that ion channels controlling the neuronal resting membrane potential (RMP) also control anesthetic sensitivity. Leak channels selective for K⁺ [ [10] , [11] , [12] and [13] ] or permeable to Na⁺ [14] are critical for establishing RMP. We hypothesized that halothane, a VA, caused immobility by altering the neuronal RMP. In C. elegans, halothane-induced immobility is acutely and completely reversed by channelrhodopsin-2 based depolarization of the RMP when expressed specifically in cholinergic neurons. Furthermore, hyperpolarizing cholinergic neurons via halorhodopsin activation increases sensitivity to halothane. The sensitivity of C. elegans to halothane can be altered by 25-fold by either manipulation of membrane conductance with optogenetic methods or generation of mutations in leak channels that set the RMP. Immobility induced by another VA, isoflurane, is not affected by these treatments, thereby excluding the possibility of nonspecific hyperactivity. The sum of our data indicates that leak channels and the RMP are important determinants of halothane-induced general anesthesia.     

Asymmetric distribution of myosin IIB in migrating endothelial cells is regulated by a rho-dependent kinase and contributes to tail retraction

All vertebrates contain two nonmuscle myosin II heavy chains, A and B, which differ in tissue expression and subcellular distributions. To understand how these distinct distributions are controlled and what role they play in cell migration, myosin IIA and IIB were examined during wound healing by bovine aortic endothelial cells. Immunofluorescence showed that myosin IIA skewed toward the front of migrating cells, coincident with actin assembly at the leading edge, whereas myosin IIB accumulated in the rear 15-30 min later. Inhibition of myosin light-chain kinase, protein kinases A, C, and G, tyrosine kinase, MAP kinase, and PIP3 kinase did not affect this asymmetric redistribution of myosin isoforms. However, posterior accumulation of myosin IIB, but not anterior distribution of myosin IIA, was inhibited by dominant-negative rhoA and by the rho-kinase inhibitor, Y-27632, which also inhibited myosin light-chain phosphorylation. This inhibition was overcome by transfecting cells with constitutively active myosin light-chain kinase. These observations indicate that asymmetry of myosin IIB, but not IIA, is regulated by light-chain phosphorylation mediated by rho-dependent kinase. Blocking this pathway inhibited tail constriction and retraction, but did not affect protrusion, suggesting that myosin IIB functions in pulling the rear of the cell forward.     

A selective difference between human Y-chromosomal DNA haplotypes

DNA analysis is making a valuable contribution to the understanding of human evolution [1]. Much attention has focused on mitochondrial DNA (mtDNA) [2] and the Y chromosome [3] and [4], both of which escape recombination and so provide information on maternal and paternal lineages, respectively. It is often assumed that the polymorphisms observed at loci on mtDNA and the Y chromosome are selectively neutral and, therefore, that existing patterns of molecular variation can be used to deduce the histories of populations in terms of drift, population movements, and cultural practices. The coalescence of the molecular phylogenies of mtDNA and the Y chromosome to recent common ancestors in Africa [5] and [6], for example, has been taken to reflect a recent origin of modern human populations in Africa. An alternative explanation, though, could be the recent selective spread of mtDNA and Y chromosome haplotypes from Africa in a population with a more complex history [7]. It is therefore important to establish whether there are selective differences between classes (haplotypes) of mtDNA and Y chromosomes and, if so, whether these differences could have been sufficient to influence the distributions of haplotypes in existing populations. A precedent for this hypothesis has been established for mtDNA in that one mtDNA background increases susceptibility to Leber hereditary optic neuropathy [8]. Although studies of nucleotide diversity in global samples of Y chromosomes have suggested an absence of recent selective sweeps or bottlenecks [9], selection may, in principle, be very important for the Y chromosome because it carries several loci affecting male fertility [10] and [11] and as many as 5% of males are infertile [11] and [12]. Here, we show that one class of infertile males, PRKX/PRKY translocation XX males, arises predominantly on a particular Y haplotypic background. Selection is, therefore, acting on Y haplotype distributions in the population.     

Recruitment of a myosin heavy chain kinase to actin-rich protrusions in Dictyostelium

Nonmuscle myosin II plays fundamental roles in cell body translocation during migration and is typically depleted or absent from actin-based cell protrusions such as lamellipodia, but the mechanisms preventing myosin II assembly in such structures have not been identified [1-3]. In Dictyostelium discoideum, myosin II filament assembly is controlled primarily through myosin heavy chain (MHC) phosphorylation. The phosphorylation of sites in the myosin tail domain by myosin heavy chain kinase A (MHCK A) drives the disassembly of myosin II filaments in vitro and in vivo [4]. To better understand the cellular regulation of MHCK A activity, and thus the regulation of myosin II filament assembly, we studied the in vivo localization of native and green fluorescent protein (GFP)-tagged MHCK A. MHCK A redistributes from the cytosol to the cell cortex in response to stimulation of Dictyostelium cells with chemoattractant in an F-actin-dependent manner. During chemotaxis, random migration, and phagocytic/endocytic events, MHCK A is recruited preferentially to actin-rich leading-edge extensions. Given the ability of MHCK A to disassemble myosin II filaments, this localization may represent a fundamental mechanism for disassembling myosin II filaments and preventing localized filament assembly at sites of actin-based protrusion.     

p116Rip targets myosin phosphatase to the actin cytoskeleton and is essential for RhoA/ROCK-regulated neuritogenesis

Activation of the RhoA-Rho kinase (ROCK) pathway stimulates actomyosin-driven contractility in many cell systems, largely through ROCK-mediated inhibition of myosin II light chain phosphatase. In neuronal cells, the RhoA-ROCK-actomyosin pathway signals cell rounding, growth cone collapse, and neurite retraction; conversely, inhibition of RhoA/ROCK promotes cell spreading and neurite outgrowth. The actin-binding protein p116(Rip), whose N-terminal region bundles F-actin in vitro, has been implicated in Rho-dependent neurite remodeling; however, its function is largely unknown. Here, we show that p116(Rip), through its C-terminal coiled-coil domain, interacts directly with the C-terminal leucine zipper of the regulatory myosin-binding subunits of myosin II phosphatase, MBS85 and MBS130. RNA interference-induced knockdown of p116(Rip) inhibits cell spreading and neurite outgrowth in response to extracellular cues, without interfering with the regulation of myosin light chain phosphorylation. We conclude that p116(Rip) is essential for neurite outgrowth and may act as a scaffold to target the myosin phosphatase complex to the actin cytoskeleton.     

Cellular Regulation of Extension and Retraction of Pseudopod-Like Blebs Produced by Nanosecond Pulsed Electric Field (nsPEF)

Recently we described a new phenomenon of anodotropic pseudopod-like blebbing in U937 cells exposed to nanosecond pulsed electric field (nsPEF). In Ca²⁺-free buffer such exposure initiates formation of pseudopod-like blebs (PLBs), protrusive cylindrical cell extensions that are distinct from apoptotic and necrotic blebs. PLBs nucleate predominantly on anode-facing cell pole and extend toward anode during nsPEF exposure. Bleb extension depends on actin polymerization and availability of actin monomers. Inhibition of intracellular Ca²⁺, cell contractility, and RhoA produced no effect on PLB initiation. Meanwhile, inhibition of WASP by wiskostatin causes dose-dependent suppression of PLB growth. Soon after the end of nsPEF exposure PLBs lose directionality of growth and then retract. Microtubule toxins nocodazole and paclitaxel did not show immediate effect on PLBs; however, nocodazole increased mobility of intracellular components during PLB extension and retraction. Retraction of PLBs is produced by myosin activation and the corresponding increase in PLB cortex contractility. Inhibition of myosin by blebbistatin reduces retraction while inhibition of RhoA–ROCK pathway by Y-27632 completely prevents retraction. Contraction of PLBs can produce cell translocation resembling active cell movement. Overall, the formation, properties, and life cycle of PLBs share common features with protrusions associated with ameboid cell migration. PLB life cycle may be controlled through activation of WASP by its upstream effectors such as Cdc42 and PIP2, and main ROCK activator—RhoA. Parallels between pseudopod-like blebbing and motility blebbing may provide new insights into their underlying mechanisms.     

A cytoskeletal demolition worker: myosin II acts as an actin depolymerization agent

Myosin II motors play several important roles in a variety of cellular processes, some of which involve active assembly/disassembly of cytoskeletal substructures. Myosin II motors have been shown to function in actin bundle turnover in neuronal growth cones and in the recycling of actin filaments during cytokinesis. Close examination had shown an intimate relationship between myosin II motor adenosine triphosphatase activity and actin turnover rate. However, the direct implication of myosin II in actin turnover is still not understood. Herein, we show, using high-resolution cryo-transmission electron microscopy, that myosin II motors control the turnover of actin bundles in a concentration-dependent manner in vitro. We demonstrate that disassembly of actin bundles occurs through two main stages: the first stage involves unbundling into individual filaments, and the second involves their subsequent depolymerization. These evidence suggest that, in addition to their “classical” contractile abilities, myosin II motors may be directly implicated in active actin depolymerization. We believe that myosin II motors may function similarly in vivo (e.g., in the disassembly of the contractile ring by fine tuning the local concentration/activity of myosin II motors).     

The RhoGEF TEM4 Regulates Endothelial Cell Migration by Suppressing Actomyosin Contractility

Persistent cellular migration requires efficient protrusion of the front of the cell, the leading edge where the actin cytoskeleton and cell-substrate adhesions undergo constant rearrangement. Rho family GTPases are essential regulators of the actin cytoskeleton and cell adhesion dynamics. Here, we examined the role of the RhoGEF TEM4, an activator of Rho family GTPases, in regulating cellular migration of endothelial cells. We found that TEM4 promotes the persistence of cellular migration by regulating the architecture of actin stress fibers and cell-substrate adhesions in protruding membranes. Furthermore, we determined that TEM4 regulates cellular migration by signaling to RhoC as suppression of RhoC expression recapitulated the loss-of-TEM4 phenotypes, and RhoC activation was impaired in TEM4-depleted cells. Finally, we showed that TEM4 and RhoC antagonize myosin II-dependent cellular contractility and the suppression of myosin II activity rescued the persistence of cellular migration of TEM4-depleted cells. Our data implicate TEM4 as an essential regulator of the actin cytoskeleton that ensures proper membrane protrusion at the leading edge of migrating cells and efficient cellular migration via suppression of actomyosin contractility.     

Acquiring "the Knowledge" of London's layout drives structural brain changes

The last decade has seen a burgeoning of reports associating brain structure with specific skills and traits (e.g., [ [1] , [2] , [3] , [4] , [5] , [6] , [7] and [8] ]). Although these cross-sectional studies are informative, cause and effect are impossible to establish without longitudinal investigation of the same individuals before and after an intervention. Several longitudinal studies have been conducted (e.g., [ [9] , [10] , [11] , [12] , [13] , [14] , [15] , [16] , [17] and [18] ]); some involved children or young adults, potentially conflating brain development with learning, most were restricted to the motor domain, and all concerned relatively short timescales (weeks or months). Here, by contrast, we utilized a unique opportunity to study average-IQ adults operating in the real world as they learned, over four years, the complex layout of London's streets while training to become licensed taxi drivers. In those who qualified, acquisition of an internal spatial representation of London was associated with a selective increase in gray matter (GM) volume in their posterior hippocampi and concomitant changes to their memory profile. No structural brain changes were observed in trainees who failed to qualify or control participants. We conclude that specific, enduring, structural brain changes in adult humans can be induced by biologically relevant behaviors engaging higher cognitive functions such as spatial memory, with significance for the “nature versus nurture” debate.