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1.  Prophase I Mouse Oocytes Are Deficient in the Ability to Respond to Fertilization by Decreasing Membrane Receptivity to Sperm and Establishing a Membrane Block to Polyspermy1 
Biology of Reproduction  2013;89(2):44.
Changes occurring as the prophase I oocyte matures to metaphase II are critical for the acquisition of competence for normal egg activation and early embryogenesis. A prophase I oocyte cannot respond to a fertilizing sperm as a metaphase II egg does, including the ability to prevent polyspermic fertilization. Studies here demonstrate that the competence for the membrane block to polyspermy is deficient in prophase I mouse oocytes. In vitro fertilization experiments using identical insemination conditions result in monospermy in 87% of zona pellucida (ZP)-free metaphase II eggs, while 92% of ZP-free prophase I oocytes have four or more fused sperm. The membrane block is associated with a postfertilization reduction in the capacity to support sperm binding, but this reduction in sperm-binding capacity is both less robust and slower to develop in fertilized prophase I oocytes. Fertilization of oocytes is dependent on the tetraspanin CD9, but little to no release of CD9 from the oocyte membrane is detected, suggesting that release of CD9-containing vesicles is not essential for fertilization. The deficiency in membrane block establishment in prophase I oocytes correlates with abnormalities in two postfertilization cytoskeletal changes: sperm-induced cortical remodeling that results in fertilization cone formation and a postfertilization increase in effective cortical tension. These data indicate that cortical maturation is a component of cytoplasmic maturation during the oocyte-to-egg transition and that the egg cortex has to be appropriately primed and tuned to be responsive to a fertilizing sperm.
The postfertilization reduction in the ability of the egg membrane to support sperm binding is less robust and slower to develop in fertilized prophase I oocytes as compared to metaphase II eggs, and this is correlated with abnormal postfertilization cytoskeletal remodeling.
PMCID: PMC4076366  PMID: 23863404
activation competence; block to polyspermy; CD9; cytoplasmic maturation; polyspermy
2.  Death and Transfiguration in Static Staphylococcus epidermidis Cultures 
PLoS ONE  2014;9(6):e100002.
The overwhelming majority of bacteria live in slime embedded microbial communities termed biofilms, which are typically adherent to a surface. However, when several Staphylococcus epidermidis strains were cultivated in static liquid cultures, macroscopic aggregates were seen floating within the broth and also sedimented at the test tube bottom. Light- and electron microscopy revealed that early-stage aggregates consisted of bacteria and extracellular matrix, organized in sheet-like structures. Perpendicular under the sheets hung a network of periodically arranged, bacteria-associated strands. During the extended cultivation, the strands of a subpopulation of aggregates developed into cross-connected wall-like structures, in which aligned bacteria formed the walls. The resulting architecture had a compartmentalized appearance. In late-stage cultures, the wall-associated bacteria disintegrated so that, henceforth, the walls were made of the coalescing remnants of lysed bacteria, while the compartment-like organization remained intact. At the same time, the majority of strand-containing aggregates with associated culturable bacteria continued to exist. These observations indicate that some strains of Staphylococcus epidermidis are able to build highly sophisticated structures, in which a subpopulation undergoes cell lysis, presumably to provide continued access to nutrients in a nutrient-limited environment, whilst maintaining structural integrity.
PMCID: PMC4070908  PMID: 24964210
3.  Depigmented-polymerised allergoids favour regulatory over effector T cells: enhancement by 1α, 25-dihydroxyvitamin D3 
BMC Immunology  2014;15:21.
Allergen immunotherapy (SIT) is the only treatment for allergic disease capable of modifying disease long term. To reduce the risk of anaphylaxis from SIT, allergen-extracts have been modified by polymerisation with glutaraldehyde to reduce IgE binding. It is suggested that these allergoid extracts also have reduced T cell activity, which could compromise clinical efficacy. Effective SIT is thought to act through regulatory T cells (Tregs) rather than activation of effector T cells. There is no published data on the activity of modified extracts on Tregs.
We compared the capacity of modified (depigmented-polymerised) versus unmodified (native) allergen extracts of grass pollen and house dust mite to stimulate proliferation/cytokine production and to modulate Treg/effector T cell frequency in cultures of peripheral blood mononuclear cells (PBMC), from volunteers sensitised to both allergens in vitro. Depigmented-polymerised allergen extracts stimulated less proliferation of PBMC, and reduced effector cell numbers after 7 days in culture than did native extracts. However, the frequency of Foxp3+ Tregs in cultures were similar to those seen with native extract so that ratios of regulatory to effector T cells were significantly increased in cultures stimulated with depigmented-polymerised extracts. Addition of 1α, 25-dihydroxyvitamin D3 further favoured Treg, and reduced effector cytokine production, but not interleukin-10.
Depigmented-polymerised allergen extracts appear to favour Treg expansion over activation of effector T cells and this may relate to their demonstrated efficacy and safety in SIT. 1α, 25-dihydroxyvitamin D3 further reduces effector T cell activation by allergen extracts and may be a useful adjuvant for SIT.
PMCID: PMC4051145  PMID: 24884430
Allergen extract; Depigmented-polymerised; Immunotherapy; Regulatory T cell; Vitamin D
4.  Molecular Mechanisms of Cellular Mechanosensing 
Nature materials  2013;12(11):10.1038/nmat3772.
Mechanical forces direct a host of cellular and tissue processes. Although much emphasis has been placed on cell-adhesion complexes as force sensors, the forces must nevertheless be transmitted through the cortical cytoskeleton. Yet how the actin cortex senses and transmits forces and how cytoskeletal proteins interact in response to the forces is poorly understood. Here, by combining molecular and mechanical experimental perturbations with theoretical multi-scale modeling, we decipher cortical mechanosensing from molecular to cellular scales. We show that forces are shared between myosin II and different actin crosslinkers, with myosin having potentiating or inhibitory effects on certain crosslinkers. Different types of cell deformations elicit distinct responses, with myosin and α-actinin responding to dilation, and filamin mainly reacting to shear. Our observations show that the accumulation kinetics of each protein may be explained by its molecular mechanisms, and that protein accumulation and the cell's viscoelastic state can explain cell contraction against mechanical load.
PMCID: PMC3838893  PMID: 24141449
5.  Chemokine responsiveness of CD4+ CD25+ regulatory and CD4+ CD25− T cells from atopic and nonatopic donors 
Allergy  2009;64(8):10.1111/j.1398-9995.2008.01962.x.
Allergic inflammation is associated with Th2-type T cells, which can be suppressed by CD4+ CD25+ regulatory T cells (Tregs). Both express chemokine receptors (CCR) 4 and CCR8, but the dynamics of expression and effect of atopic status are unknown.
To examine the expression of chemokine receptors by CD4+ CD25+ and CD4+ CD25− T cells from atopic and nonatopic donors, and document response to allergen stimulation in vitro.
Chemokine receptor expression was examined by flow cytometry and quantitative PCR of CD4+ CD25hi and CD4+ CD25− T cells from atopics and nonatopics. Responsiveness to chemokines was by actin polymerization. Dynamics of chemokine receptor expression in 6-day allergen-stimulated cultures was analysed by carboxyfluoroscein succinimidyl ester labelling.
CD4+ CD25hi Tregs preferentially expressed CCR3, CCR4, CCR5, CCR6 and CCR8. CD4+ CD25hi Tregs responded to the chemokine ligands for CCR4, CCR6 and CCR8 (CCL17, 22, 20 and 1 respectively), with no differences between atopic and nonatopic donors. Over 6-day allergen stimulation, CD4+ CD25+ T-cells downregulated CCR4 and upregulated CCR7, in contrast to CD4+ CD25− effector cells, which downregulated CCR7 and upregulated CCR4.
CCR4, CCR6 and CCR8 have potential roles in localization of both CD4+ CD25+ regulatory and CD4+ CD25− effector T cells to sites of allergic inflammation. Upregulation of CCR7 and downregulation of CCR4 upon allergen stimulation of Tregs may allow their recirculation from sites of inflammation, in contrast to retention of effector T cells.
PMCID: PMC3807784  PMID: 19208087
allergic inflammation; atopy; chemokine receptors; chemokines; regulatory T cells
6.  Asthma: T-bet – A Master Controller? 
Current biology : CB  2002;12(9):R322-R324.
The transcription factors T-bet and GATA3 are important reciprocal determinants of Th1 and Th2 T helper cell differentiation. Recent evidence suggests that these factors may affect airway immunopathology in asthma.
PMCID: PMC3807785  PMID: 12007433
7.  Depigmented Allergoids Reveal New Epitopes with Capacity to Induce IgG Blocking Antibodies 
BioMed Research International  2013;2013:284615.
Background. The synthesis of allergen-specific blocking IgGs that interact with IgE after allergen immunotherapy (SIT) has been related to clinical efficacy. The objectives were to investigate the epitope specificity of IgG-antibodies induced by depigmented-polymerized (Dpg-Pol) allergoids and unmodified allergen extracts, and examine IgE-blocking activity of induced IgG-antibodies. Methods. Rabbits were immunized with native and Dpg-Pol extracts of birch pollen, and serum samples were obtained. Recognition of linear IgG-epitopes of Bet v 1 and Bet v 2 and the capacity of these IgG-antibodies to block binding of human-IgE was determined. Results. Serum from rabbits immunized with native extracts recognised 11 linear epitopes from Bet v 1, while that from Dpg-Pol-immunized animals recognised 8. For Bet v 2, 8 epitopes were recognized by IgG from native immunized animals, and 9 from Dpg-Pol immunized one. Dpg-Pol and native immunized serum did not always recognise the same epitopes, but specific-IgG from both could block human-IgE binding sites for native extract. Conclusions. Depigmented-polymerized birch extract stimulates the synthesis of specific IgG-antibodies which recognize common but also novel epitopes compared with native extracts. IgG-antibodies induced by Dpg-Pol effectively inhibit human-IgE binding to allergens which may be part of the mechanism of action of SIT.
PMCID: PMC3816019  PMID: 24222901
8.  Cytokinesis Mechanics and Mechanosensing 
Cytoskeleton (Hoboken, N.J.)  2012;69(10):700-709.
Cytokinesis shape change occurs through the interfacing of three modules, cell mechanics, myosin II-mediated contractile stress generation and sensing, and a control system of regulatory proteins, which together ensure flexibility and robustness. This integrated system then defines the stereotypical shape changes of successful cytokinesis, which occurs under a diversity of mechanical contexts and environmental conditions.
PMCID: PMC3477504  PMID: 22761196
Actin network; Contractility; Control system; Feedback; Mechanosensing; Myosin II
9.  α-Catenin and IQGAP Regulate Myosin Localization to Control Epithelial Tube Morphogenesis in Dictyostelium 
Developmental cell  2012;23(3):533-546.
Apical actomyosin activity in animal epithelial cells influences tissue morphology, and drives morphogenetic movements during development. The molecular mechanisms leading to myosin II accumulation at the apical membrane and its exclusion from other membranes are poorly understood. We show that in the non-metazoan Dictyostelium discoideum, myosin II localizes apically in tip epithelial cells that surround the stalk, and constriction of this epithelial tube is required for proper morphogenesis. IQGAP1 and its binding partner cortexillin I function downstream of α- and β-catenin to exclude myosin II from the basolateral cortex and promote apical accumulation of myosin II. Deletion of IQGAP1 or cortexillin compromises epithelial morphogenesis without affecting cell polarity. These results reveal that apical localization of myosin II is a conserved morphogenetic mechanism from non-metazoans to vertebrates, and identify a hierarchy of proteins that regulate the polarity and organization of an epithelial tube in a simple model organism.
PMCID: PMC3443284  PMID: 22902739
10.  A Summer Academic Research Experience for Disadvantaged Youth 
CBE Life Sciences Education  2013;12(3):410-418.
We describe an outreach initiative to provide disadvantaged youth with an intensive academic research experience. To offer an effective internship for these underrepresented youth, one needs to develop a comprehensive program that addresses the students' academic, professional, and personal needs.
Internships are an effective way of connecting high school students in a meaningful manner to the sciences. Disadvantaged minorities have fewer opportunities to participate in internships, and are underrepresented in both science, technology, engineering, and mathematics majors and careers. We have developed a Summer Academic Research Experience (SARE) program that provides an enriching academic internship to underrepresented youth. Our program has shown that to have a successful internship for these disadvantaged youth, several issues need to be addressed in addition to scientific mentoring. We have found that it is necessary to remediate and/or fortify basic academic skills for students to be successful. In addition, students need to be actively coached in the development of professional skills, habits, and attitudes necessary for success in the workplace. With all these factors in place, these youths can become better students, compete on a more level playing field in their internships, and increase their potential of participating actively in the sciences in the future.
PMCID: PMC3763009  PMID: 24006390
11.  Separation anxiety: stress, tension and cytokinesis 
Experimental Cell Research  2012;318(12):1428-1434.
Cytokinesis, the physical separation of a mother cell into two daughter cells, progresses through a series of well-defined changes in morphology. These changes involve distinct biochemical and mechanical processes. Here, we review the mechanical features of cells during cytokinesis, discussing both the material properties as well as sources of stresses, both active and passive, which lead to the observed changes in morphology. We also describe a mechanosensory feedback control system that regulates protein localization and shape progression during cytokinesis.
PMCID: PMC3372636  PMID: 22487096
Actin dynamics; cell division; cleavage furrow; mechanosensation; myosin II
12.  Bringing the physical sciences into your cell biology research 
Molecular Biology of the Cell  2012;23(21):4167-4170.
Historically, much of biology was studied by physicists and mathematicians. With the advent of modern molecular biology, a wave of researchers became trained in a new scientific discipline filled with the language of genes, mutants, and the central dogma. These new molecular approaches have provided volumes of information on biomolecules and molecular pathways from the cellular to the organismal level. The challenge now is to determine how this seemingly endless list of components works together to promote the healthy function of complex living systems. This effort requires an interdisciplinary approach by investigators from both the biological and the physical sciences.
PMCID: PMC3484095  PMID: 23112230
13.  The spatial and mechanical challenges of female meiosis 
Molecular reproduction and development  2011;78(10-11):769-777.
Recent work shows that cytokinesis and other cellular morphogenesis events are tuned by an interplay among biochemical signals, cell shape, and cellular mechanics. In cytokinesis, this includes cross-talk between the cortical cytoskeleton and the mitotic spindle in coordination with cell cycle control, resulting in characteristic changes in cellular morphology and mechanics through metaphase and cytokinesis. The changes in cellular mechanics affect not just overall cell shape, but also mitotic spindle morphology and function. This review will address how these principles apply to oocytes undergoing the asymmetric cell divisions of meiosis I and II. The biochemical signals that regulate cell cycle timing during meiotic maturation and egg activation are crucial for temporal control of meiosis. Spatial control of the meiotic divisions is also important, ensuring that the chromosomes are segregated evenly and that meiotic division is clearly asymmetric, yielding two daughter cells – oocyte and polar body – with enormous volume differences. In contrast to mitotic cells, the oocyte does not undergo overt changes in cell shape with its progression through meiosis, but instead maintains a relatively round morphology with the exception of very localized changes at the time of polar body emission. Placement of the metaphase-I and -II spindles at the oocyte periphery is clearly important for normal polar body emission, although this is likely not the only control element. Here, consideration is given to how cellular mechanics could contribute to successful mammalian female meiosis, ultimately affecting egg quality and competence to form a healthy embryo.
PMCID: PMC3196790  PMID: 21774026
oocyte; fertilization; meiotic maturation; cytokinesis; cell cortex; cytoskeleton; spindle; myosin II; ezrin-radixin-moesin
14.  CD4+CD25+ regulatory T cells reverse established allergic airway inflammation and prevent airway remodeling 
CD4+CD25+ regulatory T cells can inhibit excessive T-cell responses in vivo. We have previously demonstrated that prophylactic administration of CD4+CD25+ regulatory T cells suppresses the development of acute allergen-induced airway inflammation in vivo.
We sought to determine the effect of therapeutic transfer of CD4+CD25+ regulatory T cells on established pulmonary inflammation and the subsequent development of airway remodeling.
CD4+CD25+ cells were transferred after the onset of allergic inflammation, and airway challenges were continued to induce chronic inflammation and airway remodeling.
Administration of CD4+CD25+ regulatory T cells reduced established lung eosinophilia, TH2 infiltration, and expression of IL-5, IL-13, and TGF-β. Moreover, subsequent mucus hypersecretion and peribronchial collagen deposition were reduced after prolonged challenge. In contrast, transfer of CD4+CD25+ regulatory T cells had no effect on established airway hyperreactivity either 7 days or 4 weeks after transfer.
In this study we demonstrate for the first time that therapeutic transfer of CD4+CD25+ regulatory T cells can resolve features of chronic allergen-induced inflammation and prevent development of airway remodeling.
PMCID: PMC3389733  PMID: 18672278
Regulatory T cells; TH2 cells; eosinophils; TGF-β; airway remodeling; allergic inflammation
15.  A mechanosensory system governs myosin II accumulation in dividing cells 
Molecular Biology of the Cell  2012;23(8):1510-1523.
A mechanosensory system is characterized that fine-tunes the level of myosin II at the cleavage furrow. This mechanosensory system consists of the mechanosensory module composed of myosin II and cortexillin I and a mechanotransduction loop that includes IQGAP2, kinesin-6, and INCENP.
The mitotic spindle is generally considered the initiator of furrow ingression. However, recent studies suggest that furrows can form without spindles, particularly during asymmetric cell division. In Dictyostelium, the mechanoenzyme myosin II and the actin cross-linker cortexillin I form a mechanosensor that responds to mechanical stress, which could account for spindle-independent contractile protein recruitment. Here we show that the regulatory and contractility network composed of myosin II, cortexillin I, IQGAP2, kinesin-6 (kif12), and inner centromeric protein (INCENP) is a mechanical stress–responsive system. Myosin II and cortexillin I form the core mechanosensor, and mechanotransduction is mediated by IQGAP2 to kif12 and INCENP. In addition, IQGAP2 is antagonized by IQGAP1 to modulate the mechanoresponsiveness of the system, suggesting a possible mechanism for discriminating between mechanical and biochemical inputs. Furthermore, IQGAP2 is important for maintaining spindle morphology and kif12 and myosin II cleavage furrow recruitment. Cortexillin II is not directly involved in myosin II mechanosensitive accumulation, but without cortexillin I, cortexillin II's role in membrane–cortex attachment is revealed. Finally, the mitotic spindle is dispensable for the system. Overall, this mechanosensory system is structured like a control system characterized by mechanochemical feedback loops that regulate myosin II localization at sites of mechanical stress and the cleavage furrow.
PMCID: PMC3327329  PMID: 22379107
16.  A Novel Role for Aquaporin-5 in Enhancing Microtubule Organization and Stability 
PLoS ONE  2012;7(6):e38717.
Aquaporin-5 (AQP5) is a water-specific channel located on the apical surface of airway epithelial cells. In addition to regulating transcellular water permeability, AQP5 can regulate paracellular permeability, though the mechanisms by which this occurs have not been determined. Microtubules also regulate paracellular permeability. Here, we report that AQP5 promotes microtubule assembly and helps maintain the assembled microtubule steady state levels with slower turnover dynamics in cells. Specifically, reduced levels of AQP5 correlated with lower levels of assembled microtubules and decreased paracellular permeability. In contrast, overexpression of AQP5 increased assembly of microtubules, with evidence of increased MT stability, and promoted the formation of long straight microtubules in the apical domain of the epithelial cells. These findings indicate that AQP5-mediated regulation of microtubule dynamics modulates airway epithelial barrier properties and epithelial function.
PMCID: PMC3371026  PMID: 22715407
17.  Deconvolution of the Cellular Force-Generating Subsystems that Govern Cytokinesis Furrow Ingression 
PLoS Computational Biology  2012;8(4):e1002467.
Cytokinesis occurs through the coordinated action of several biochemically-mediated stresses acting on the cytoskeleton. Here, we develop a computational model of cellular mechanics, and using a large number of experimentally measured biophysical parameters, we simulate cell division under a number of different scenarios. We demonstrate that traction-mediated protrusive forces or contractile forces due to myosin II are sufficient to initiate furrow ingression. Furthermore, we show that passive forces due to the cell's cortical tension and surface curvature allow the furrow to complete ingression. We compare quantitatively the furrow thinning trajectories obtained from simulation with those observed experimentally in both wild-type and myosin II null Dictyostelium cells. Our simulations highlight the relative contributions of different biomechanical subsystems to cell shape progression during cell division.
Author Summary
Cytokinesis, the physical separation of a mother cell into two daughter cells, requires force to deform the cell. Though there is ample evidence in many systems that myosin II provides some of this force, it is also well known that some cell types can divide in the absence of myosin II. To elucidate the mechanisms by which cells control furrow ingression, we developed a computational model of cellular dynamics during cytokinesis in the social amoeba, Dictyostelium discoideum. We took advantage of a large number of experimentally measured parameters and well-characterized furrow ingression dynamics for a number of different strains. Our simulations demonstrate that there are distinct phases of cytokinesis. Myosin II plays a role providing the stress that initiates furrow ingression. In its absence, however, this force can be supplied by a combination of adhesion and protrusion-mediated stresses. Thereafter, Laplace-like pressures take over and provide stresses that enable the cell to divide. Overall, we show how various mechanical parameters quantitatively impact furrow ingression kinetics, accounting for the cytokinesis dynamics of wild type and mutant cell-lines.
PMCID: PMC3343096  PMID: 22570593
18.  Cytokinesis through biochemical-mechanical feedback loops 
Cytokinesis is emerging as a control system defined by interacting biochemical and mechanical modules, which form a system of feedback loops. This integrated system accounts for the regulation and kinetics of cytokinesis furrowing and demonstrates that cytokinesis is a whole-cell process in which the global and equatorial cortices and cytoplasm are active players in the system. Though originally defined in Dictyostelium, features of the control system are recognizable in other organisms, suggesting a universal mechanism for cytokinesis regulation and contractility.
PMCID: PMC2991468  PMID: 20709619
Actin; Cell Mechanics; Control System; Cytokinesis; Mechanosensing; Myosin II
19.  14-3-3 coordinates microtubules, Rac, and myosin II to control cell mechanics and cytokinesis 
Current biology : CB  2010;20(21):1881-1889.
During cytokinesis, regulatory signals are presumed to emanate from the mitotic spindle. However, what these signals are and how they lead to the spatiotemporal changes in the cortex structure, mechanics, and regional contractility are not well understood in any system.
To investigate pathways that link the microtubule network to the cortical changes that promote cytokinesis, we used chemical genetics in Dictyostelium to identify genetic suppressors of nocodazole, a microtubule depolymerizer. We identified 14-3-3 and found that it is enriched in the cortex, helps maintain steady state microtubule length, contributes to normal cortical tension, modulates actin wave formation, and controls the symmetry and kinetics of cleavage furrow contractility during cytokinesis. Furthermore, 14-3-3 acts downstream of a Rac small GTPase (RacE), associates with myosin II heavy chain and is needed to promote myosin II bipolar thick filament remodeling.
14-3-3 connects microtubules, Rac and myosin II to control several aspects of cortical dynamics, mechanics, and cytokinesis cell shape change. Further, 14-3-3 interacts directly with myosin II heavy chain to promote bipolar thick filament remodeling and distribution. Overall, 14-3-3 appears to integrate several critical cytoskeletal elements that drive two important processes cytokinesis shape change and cell mechanics.
PMCID: PMC2975807  PMID: 20951045
20.  Dictyostelium huntingtin controls chemotaxis and cytokinesis through the regulation of myosin II phosphorylation 
Molecular Biology of the Cell  2011;22(13):2270-2281.
This work shows that huntingtin protein (Htt) regulates the phosphorylation status of myosin II during chemotaxis and cytokinesis through protein phosphatase 2A (PP2A). Our findings provide novel insights into the physiological function of Htt and the pathogenesis of Huntington's disease.
Abnormalities in the huntingtin protein (Htt) are associated with Huntington's disease. Despite its importance, the function of Htt is largely unknown. We show that Htt is required for normal chemotaxis and cytokinesis in Dictyostelium discoideum. Cells lacking Htt showed slower migration toward the chemoattractant cAMP and contained lower levels of cortical myosin II, which is likely due to defects in dephosphorylation of myosin II mediated by protein phosphatase 2A (PP2A). htt− cells also failed to maintain myosin II in the cortex of the cleavage furrow, generating unseparated daughter cells connected through a thin cytoplasmic bridge. Furthermore, similar to Dictyostelium htt− cells, siRNA-mediated knockdown of human HTT also decreased the PP2A activity in HeLa cells. Our data indicate that Htt regulates the phosphorylation status of myosin II during chemotaxis and cytokinesis through PP2A.
PMCID: PMC3128529  PMID: 21562226
21.  Cortical Mechanics and Meiosis II Completion in Mammalian Oocytes Are Mediated by Myosin-II and Ezrin-Radixin-Moesin (ERM) Proteins 
Molecular Biology of the Cell  2010;21(18):3182-3192.
Analysis of mouse oocyte mechanics shows that effective tension drops 6-fold from prophase I to metaphase II; the metaphase II egg has a 2.5-fold tension differential between the cortex over the spindle and the opposite cortex. Manipulation of actin, myosin-II, or ERMs alters tension levels and induces spindle abnormalities during meiosis II.
Cell division is inherently mechanical, with cell mechanics being a critical determinant governing the cell shape changes that accompany progression through the cell cycle. The mechanical properties of symmetrically dividing mitotic cells have been well characterized, whereas the contribution of cellular mechanics to the strikingly asymmetric divisions of female meiosis is very poorly understood. Progression of the mammalian oocyte through meiosis involves remodeling of the cortex and proper orientation of the meiotic spindle, and thus we hypothesized that cortical tension and stiffness would change through meiotic maturation and fertilization to facilitate and/or direct cellular remodeling. This work shows that tension in mouse oocytes drops about sixfold during meiotic maturation from prophase I to metaphase II and then increases ∼1.6-fold upon fertilization. The metaphase II egg is polarized, with tension differing ∼2.5-fold between the cortex over the meiotic spindle and the opposite cortex, suggesting that meiotic maturation is accompanied by assembly of a cortical domain with stiffer mechanics as part of the process to achieve asymmetric cytokinesis. We further demonstrate that actin, myosin-II, and the ERM (Ezrin/Radixin/Moesin) family of proteins are enriched in complementary cortical domains and mediate cellular mechanics in mammalian eggs. Manipulation of actin, myosin-II, and ERM function alters tension levels and also is associated with dramatic spindle abnormalities with completion of meiosis II after fertilization. Thus, myosin-II and ERM proteins modulate mechanical properties in oocytes, contributing to cell polarity and to completion of meiosis.
PMCID: PMC2938384  PMID: 20660156
22.  14-3-3, an integrator of cell mechanics and cytokinesis 
Small GTPases  2010;1(3):165-169.
One of the goals of understanding cytokinesis is to uncover the molecular regulation of the cellular mechanical properties that drive cell shape change. Such regulatory pathways are likely to be used at multiple stages of a cell's life, but are highly featured during cell division. Recently, we demonstrated that 14-3-3 (encoded by a single gene in the social amoeba Dictyostelium discoideum) serves to integrate key cytoskeletal components—microtubules, Rac and myosin II—to control cell mechanics and cytokinesis. As 14-3-3 proteins are frequently altered in a variety of human tumors, we extend these observations to suggest possible additional roles for how 14-3-3 proteins may contribute to tumorigenesis.
PMCID: PMC3116603  PMID: 21686271
14-3-3; bipolar thick filament; cancer; cell mechanics; cortical tension; cytokinesis; microtubules; myosin II; Rac; tumorigenesis; tumor suppressor
23.  Mechanosensing through Cooperative Interactions between Myosin-II and the Actin Crosslinker Cortexillin-I 
Current biology : CB  2009;19(17):1421-1428.
Mechanosensing governs many processes from molecular to organismal levels, including during cytokinesis where it ensures successful and symmetrical cell division. While many proteins are now known to be force sensitive, myosin motors with their ATPase activity and force-sensitive mechanical steps are well poised to facilitate cellular mechanosensing. For a myosin motor to experience tension, the actin filament must also be anchored.
Here, we find a cooperative relationship between myosin-II and the actin crosslinker cortexillin-I where both proteins are essential for cellular mechanosensory responses. While many functions of cortexillin-I and myosin-II are dispensable for cytokinesis, all are required for full mechanosensing. Our analysis demonstrates that this mechanosensor has three critical elements: the myosin motor where the lever arm acts as a force amplifier, a force-sensitive bipolar thick filament assembly, and a long lived actin crosslinker, which anchors the actin filament so that the motor may experience tension. We also demonstrate that a Rac small GTPase inhibits this mechanosensory module during interphase, allowing the module to be primarily active during cytokinesis.
Overall, myosin-II and cortexillin-I define a cellular-scale mechanosensor that controls cell shape during cytokinesis. This system is exquisitely tuned through the enzymatic properties of the myosin motor, its lever arm length and bipolar thick filament assembly dynamics. The system also requires cortexillin-I to stably anchor the actin filament so that the myosin motor can experience tension. Through this cross-talk, myosin-II and cortexillin-I define a cellular-scale mechanosensor that monitors and corrects shape defects, ensuring symmetrical cell division.
PMCID: PMC2763054  PMID: 19646871
24.  Involvement of the Cytoskeleton in Controlling Leading-Edge Function during Chemotaxis 
Molecular Biology of the Cell  2010;21(11):1810-1824.
Cells activate signaling pathways at the site closest to the chemoattractant source that lead to pseudopod formation and directional movement up the gradient. We demonstrate that cytoskeletal components required for cortical tension, including MyoII and IQGAP/cortexillins help regulate the level and timing of leading-edge pathways.
In response to directional stimulation by a chemoattractant, cells rapidly activate a series of signaling pathways at the site closest to the chemoattractant source that leads to F-actin polymerization, pseudopod formation, and directional movement up the gradient. Ras proteins are major regulators of chemotaxis in Dictyostelium; they are activated at the leading edge, are required for chemoattractant-mediated activation of PI3K and TORC2, and are one of the most rapid responders, with activity peaking at ∼3 s after stimulation. We demonstrate that in myosin II (MyoII) null cells, Ras activation is highly extended and is not restricted to the site closest to the chemoattractant source. This causes elevated, extended, and spatially misregulated activation of PI3K and TORC2 and their effectors Akt/PKB and PKBR1, as well as elevated F-actin polymerization. We further demonstrate that disruption of specific IQGAP/cortexillin complexes, which also regulate cortical mechanics, causes extended activation of PI3K and Akt/PKB but not Ras activation. Our findings suggest that MyoII and IQGAP/cortexillin play key roles in spatially and temporally regulating leading-edge activity and, through this, the ability of cells to restrict the site of pseudopod formation.
PMCID: PMC2877640  PMID: 20375144
25.  Automated characterization of cell shape changes during amoeboid motility by skeletonization 
BMC Systems Biology  2010;4:33.
The ability of a cell to change shape is crucial for the proper function of many cellular processes, including cell migration. One type of cell migration, referred to as amoeboid motility, involves alternating cycles of morphological expansion and retraction. Traditionally, this process has been characterized by a number of parameters providing global information about shape changes, which are insufficient to distinguish phenotypes based on local pseudopodial activities that typify amoeboid motility.
We developed a method that automatically detects and characterizes pseudopodial behavior of cells. The method uses skeletonization, a technique from morphological image processing to reduce a shape into a series of connected lines. It involves a series of automatic algorithms including image segmentation, boundary smoothing, skeletonization and branch pruning, and takes into account the cell shape changes between successive frames to detect protrusion and retraction activities. In addition, the activities are clustered into different groups, each representing the protruding and retracting history of an individual pseudopod.
We illustrate the algorithms on movies of chemotaxing Dictyostelium cells and show that our method makes it possible to capture the spatial and temporal dynamics as well as the stochastic features of the pseudopodial behavior. Thus, the method provides a powerful tool for investigating amoeboid motility.
PMCID: PMC2864235  PMID: 20334652

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