Validation of a biodynamic model of pushing and pulling.

Pushing and pulling during manual material handling can increase the compressive forces on the lumbar disc region while creating high shear forces at the shoe-floor interface. A sagittal plane dynamic model derived from previous biomechanical models was developed to predict L5/S1 compressive force and required coefficients of friction during dynamic cart pushing and pulling. Before these predictions could be interpreted, however, it was necessary to validate model predictions against independently measured values of comparable quantities. This experiment used subjects of disparate stature and body mass, while task factors such as cart resistance and walking speed were varied. Predicted ground reaction forces were compared with those measured by a force platform, with correlations up to 0.67. Predicted erector spinae and rectus abdominus muscle forces were compared with muscle forces derived from RMS-EMGs of the respective muscle groups, using a static force build-up regression relationship to transform the dynamic RMS-EMGs to trunk muscle forces. Although correlations were low, this was attributed in part to the use of surface EMG on subjects of widely varied body mass. The biodynamic model holds promise as a tool for analysis of actual industrial pushing and pulling tasks, when carefully applied.     

The pulling,pushing and fusing of lens fibers

Lens development and differentiation are intricate and complex processes characterized by distinct molecular and morphological changes. The growth of a transparent lens involves proliferation of the epithelial cells and their subsequent differentiation into secondary fiber cells. Prior to differentiation, epithelial cells at the lens equator exit from the cell cycle and elongate into long, ribbon-like cells. Fiber cell elongation takes place bidirectionally as fiber tips migrate both anteriorly and posteriorly along the apical surface of the epithelium and inner surface of the capsule, respectively. The differentiating fiber cells move inward from the periphery to the center of the lens on a continuous basis as the lens grows throughout life. Finally, when fiber cells reach the center or suture line, their basal and apical tips detach from the epithelium and capsule, respectively, and interlock with cells from the opposite direction of the lens and form the suture line. Further, symmetric packing of fiber cells and degradation of most of the cellular organelle during fiber cell terminal differentiation are crucial for lens transparency. These sequential events are presumed to depend on cytoskeletal dynamics and cell adhesive interactions; however, our knowledge of regulation of lens fiber cell cytosketal reorganization, cell adhesive interactions and mechanotransduction, and their role in lens morphogenesis and function is limited at present. Recent biochemical and molecular studies have targeted cytoskeletal signaling proteins, including Rho GTPases, Abl kinase interacting proteins, cell adhesion molecules, myosin II, Src kinase and phosphoinositide 3-kinase in the developing chicken and mouse lens and characterized components of the fiber cell basal membrane complex. These studies have begun to unravel the vital role of cytoskeletal proteins and their regulatory pathways in control of lens morphogenesis, fiber cell elongation, migration, differentiation, survival and mechanical properties.     

Postural differences in shoulder dynamics during pushing and pulling

Assessments of shoulder dynamics (e.g. the inertial, viscous, and stiffness properties of the joint) can provide important insights into the stability of the joint at rest and during volitional contraction. The purpose of this study was to investigate how arm posture influences shoulder dynamics while generating pushing or pulling torques in the horizontal plane. Sixteen healthy participants were examined in seven postures encompassing a large workspace of the shoulder. At each posture, the participant’s shoulder was rapidly perturbed while measuring the resultant change in shoulder torque about the glenohumeral axis. Participants were examined both at rest and while producing horizontal flexion and extension torques scaled to 15% of a maximum voluntary contraction. Shoulder stiffness, viscosity, and damping ratio were estimated using impedance-based matching, and changes in these outcome measures with torque level, elevation angle, and plane of elevation angle were explored with a linear mixed effects model. Shoulder stiffness was found to decrease with increasing elevation angles (p < 0.001) without subsequent changes in viscosity, leading to a greater damping ratios at higher elevation angles (p < 0.001). Shoulder stiffness, viscosity, and damping ratio (all p < 0.05) were all found to significantly increase as the plane of elevation of the arm was increased. The relationship between the viscosity, stiffness and the damping ratio of the shoulder is one that the central nervous system must regulate in order to maintain stability, protect against injury, and control the shoulder joint as the inertial and muscle contributions change across different arm postures.     

Metabolic engineering of the oleaginous alga Nannochloropsis for enriching eicosapentaenoic acid in triacylglycerol by combined pulling and pushing strategies

The marine alga Nannochloropsis oceanica has been considered as a promising photosynthetic cell factory for synthesizing eicosapentaenoic acid (EPA), yet the accumulation of EPA in triacylglycerol (TAG) is restricted to an extreme low level. Poor channeling of EPA to TAG was observed in N. oceanica under TAG induction conditions, likely due to the weak activity of endogenous diacylglycerol acyltransferases (DGATs) on EPA-CoA. Screening over thirty algal DGATs revealed potent enzymes acting on EPA-CoA. Whilst overexpressing endogenous DGATs had no or slight effect on EPA abundance in TAG, introducing selected DGATs with strong activity on EPA-CoA, particularly the Chlamydomonas-derived CrDGTT1, which resided at the outermost membrane of the chloroplast and provided a strong pulling power to divert EPA to TAG for storage and protection, led to drastic increases in EPA abundance in TAG and TAG-derived EPA level in N. oceanica. They were further promoted by additional overexpression of an elongase gene involved in EPA biosynthesis, reaching 5.9- and 12.3-fold greater than the control strain, respectively. Our results together demonstrate the concept of applying combined pulling and pushing strategies to enrich EPA in algal TAG and provide clues for the enrichment of other desired fatty acids in TAG as well.     

Polymer motors: pushing out the front and pulling up the back

Mechanical work in cells is performed by specialized motor proteins that operate in a continuous mechanochemical cycle. Less complex, but still efficient, 'one-shot' motors evolved based on the assembly and disassembly of polymers. We review the mechanisms of pushing and pulling by actin and microtubule filaments and the organizational principles of actin networks. We show how these polymer force generators are used for the propulsion of intracellular pathogens, protrusion of lamellipodia and mitotic movements. We discuss several examples of cellular forces generated by the assembly and disassembly of polymer gels.     

Finding the cell center by a balance of dynein and myosin pulling and microtubule pushing: a computational study

The centrosome position in many types of interphase cells is actively maintained in the cell center. Our previous work indicated that the centrosome is kept at the center by pulling force generated by dynein and actin flow produced by myosin contraction and that an unidentified factor that depends on microtubule dynamics destabilizes position of the centrosome. Here, we use modeling to simulate the centrosome positioning based on the idea that the balance of three forces-dyneins pulling along microtubule length, myosin-powered centripetal drag, and microtubules pushing on organelles-is responsible for the centrosome displacement. By comparing numerical predictions with centrosome behavior in wild-type and perturbed interphase cells, we rule out several plausible hypotheses about the nature of the microtubule-based force. We conclude that strong dynein- and weaker myosin-generated forces pull the microtubules inward competing with microtubule plus-ends pushing the microtubule aster outward and that the balance of these forces positions the centrosome at the cell center. The model also predicts that kinesin action could be another outward-pushing force. Simulations demonstrate that the force-balance centering mechanism is robust yet versatile. We use the experimental observations to reverse engineer the characteristic forces and centrosome mobility.     

The pulling,pushing and fusing of lens fibers: A role for Rho GTPases

Lens development and differentiation are intricate and complex processes characterized by distinct molecular and morphological changes. The growth of a transparent lens involves proliferation of the epithelial cells and their subsequent differentiation into secondary fiber cells. Prior to differentiation, epithelial cells at the lens equator exit from the cell cycle and elongate into long, ribbon-like cells. Fiber cell elongation takes place bidirectionally as fiber tips migrate both anteriorly and posteriorly along the apical surface of the epithelium and inner surface of the capsule, respectively. The differentiating fiber cells move inward from the periphery to the center of the lens on a continuous basis as the lens grows throughout life. Finally, when fiber cells reach the center or suture line, their basal and apical tips detach from the epithelium and capsule, respectively, and interlock with cells from the opposite direction of the lens and form the suture line. Further, symmetric packing of fiber cells and degradation of most of the cellular organelle during fiber cell terminal differentiation are crucial for lens transparency. These sequential events are presumed to depend on cytoskeletal dynamics and cell adhesive interactions; however, our knowledge of regulation of lens fiber cell cytosketal reorganization, cell adhesive interactions and mechanotransduction, and their role in lens morphogenesis and function is limited at present. Recent biochemical and molecular studies have targeted cytoskeletal signaling proteins, including Rho GTPases, Abl kinase interacting proteins, cell adhesion molecules, myosin II, Src kinase and phosphoinositide 3-kinase in the developing chicken and mouse lens and characterized components of the fiber cell basal membrane complex. These studies have begun to unravel the vital role of cytoskeletal proteins and their regulatory pathways in control of lens morphogenesis, fiber cell elongation, migration, differentiation, survival and mechanical properties.Key words: lens, fiber cells, elongation, migration, adhesion, Rho GTPasesLens morphogenesis involves a complex network of regulatory genes and interplay between growth factor, mitogenic, cell adhesive and cytoskeletal signaling pathways. The lens originates from surface ectoderm near the optic vesicle and lens vesicle that is formed via invagination of lens placode differentiates into primary fibers (the posterior half ) and epithelial cells (the anterior half ). These changes in embryonic cells control the lens distinctive anterior-posterior polarity. Subsequently, the lens grows through the proliferation of epithelial cells and the differentiation of their progeny into secondary fiber cells.^1,2 The continuous addition of new fiber cells at the lens periphery leads to a gradual inward movement of older cells to the center of the lens. The ectodermal basement membrane that surrounds the lens vesicle thickens to form the lens capsule and is composed of mainly proteins of extracellular matrix.^2,3 Since the lens does not shed cells, they are retained throughout the lens''s life and are packed symmetrically within the lens⁴ (Fig. 1).Open in a separate windowFigure 1Diagram of organization of lens epithelial and differentiating fiber cells. The lens is enclosed by a thick capsule consisting of various extracellular matrix proteins. Lens epithelial cells at the equator divide and exit from the cell cycle, and as they exit from the cell cycle, they start to elongate bidirectionally by making apical (AMC) and basal (BMC) membrane complexes with epithelium and capsule, respectively. As fiber cells elongate, they are pushed down and migrate toward the center. As the fiber cells migrate toward the center, both the basal and apical membrane complexes are expected to undergo changes in a regulated manner to control fiber cell adhesive, protrusive and contractile activity. Finally, when the fiber cells reach the center or suture line, their basal and apical ends detach from the epithelium and capsule, respectively and interlock with cells from the opposite direction of the lens and form suture. During fiber cell elongation and differentiation, cell adhesive interactions are reorganized extensively, and terminally differentiated fiber cells exhibit loss of cellular organelle and extensive membrane remodeling with unique ball and socket interdigitations. Arrows indicate the direction of fiber cell movement. This schematic is a modified version of Figure 2 from Lovicu and McAvoy.¹Lens fiber cell elongation and differentiation is associated with a remarkable change in cell morphology, with the length of fiber cells increasing on the order of several hundredfold. These morphological changes are associated with extensive membrane and cortical cytoskeletal remodeling, actomyosin reorganization and cell adhesion turnover.^5–17 Additionally, the tips of the elongating fiber cells at both the anterior and posterior terminals slide along the lens epithelium and capsule, respectively, as these cells migrate inward, and finally detach at the suture, where they form contacts with their counterparts from the opposite side of the lens.^4,12 These cell movements are fundamental for maintaining distinct lens fiber cell polarity and are temporally and spatially regulated as the lens grows continuously throughout life.^1,2,12 Another unique feature of the lens is that during fiber cell terminal differentiation, all the cellular organelles, including nuclei, endoplasmic reticulum and mitochondria, are degraded in a programmed manner.¹⁸ It has been well documented that lens epithelial cell elongation and differentiation is associated with reorganization of actin cytoskeleton, increased ratio of G-actin to F-actin, integrin switching, formation of N-cadherin linked cell adhesions, and expression of actin capping protein tropomodulin.^{5,6,9,10,13,15,17,19–21} Importantly, disruption of actin cytoskeletal organization has been shown to impair lens epithelial differentiation and induce cataract formation, indicating the significance of actin cytoskeleton in lens differentiation and maintenance of lens optical quality.^14,22 Further, during accommodation, lens shape is changed in a reversible manner. Therefore, the tensional homeostasis between actomyosin inside the fiber cell and fiber cell adhesion on the inner side of the lens capsule is considered to be crucial for accommodation.¹²In the developing mouse and chicken lens, the tips of the fiber cells (both apical and basal) have been reported to cluster with different cytoskeletal proteins, including actin, myosin II, actin capping protein tropomodulin, and N-cadherins.^10,19,21 Similarly, adhesion regulating signaling molecules including integrins, focal adhesion kinase, Cdk5, abl kinase interacting protein (Abi-2), and Rho GTPases have been shown to localize to the fiber cell apical and basal tips.^20,23–26 Moreover, isolation and characterization of the fiber cell basal membrane complexes (BMCs) had revealed a symmetric organization of N-cadherin, myosin II, actin in association with myosin light chain kinase, focal adhesion kinase, β1 integrin and caldesmon.¹² The signaling activity, tensional property and dynamics of BMCs are thought to control the coordinated migration of fiber cells along the lens capsule, formation of lens suture line, and lens accommodation.¹² Additionally, the BMCs have been shown to undergo a characteristic regional rearrangement (including size and shape) during lens elongation and migration along the lens capsule.²⁷ Therefore, impaired fiber cell migration on the lens capsule is expected to induce cataractogenesis.²⁷ Taken together, these different observations convincingly indicate the importance of cytoskeleton and cell adhesion regulatory mechanisms in lens fiber cell elongation and migration.Although important insights have emerged regarding external cues controlling lens epithelial cell proliferation, elongation and differentiation, little is known regarding the specific signaling pathways that drive the processes culminating in fiber cell formation, migration, packing and maturation.^1,7,28 For example, growth factors are known to play key roles in influencing cell fates during development. Some of the major growth factor families, including FGFs and TGFβ/BMPs, have been shown to be involved in the regulation of lens developmental processes and primary fiber cell differentiation via ERK kinase activation.^1,28,29 However, the identity and role of signaling pathways acting downstream to growth factors regulating lens secondary fiber cell elongation, migration, adhesion, membrane remodeling and survival are poorly understood.^1,12,21,30 In particular, regulatory mechanisms involved in cytoskeletal reorganization, tensional force and cell adhesive interactions during these cellular processes have yet be identified and characterized.^{7,9,12,21,30–32}Our laboratory has been working on a broad hypothesis that the actin cytoskeletal and cell adhesive signaling mechanisms composed of Rho GTPases (Rho, Rac and Cdc42) and their effector molecules play a critical role in controlling lens growth and differentiation, and in maintaining lens integrity.⁷ The Rho family of small GTPases regulates morphogenesis, polarity, migration and cell adhesion.³³ These proteins bind GTP, exhibit GTPase activity, and cycle between an inactive GDP-bound form and an active GTP-bound form. This cycling is regulated by three groups of proteins: guanine-nucleotide exchange factors, which facilitate the exchange of GDP for GTP, thus rendering Rho GTPases active; GTPase-activating proteins, which regulate the inactivation of Rho by accelerating intrinsic GTPase activity and converting Rho GTPases back to their GDP-bound form; and GDP dissociation inhibitors (GDIs), which inhibit the dissociation of GDP bound to Rho GTPases.^33,34 The GTP-bound form of the Rho GTPases interact with downstream effectors, which include protein kinases (e.g., ROCK and PAK), regulators of actin polymerization (e.g., N-WASP/WAVE, PI3-kinase and mDia), and other proteins with adaptor functions.³³ The selective interaction of the different Rho GTPases with a variety of effectors determines the final outcome of their activation.³³ For example, during cell movement, Rac and Cdc42 stimulate formation of protrusions at the leading edges of cells, and RhoA induces retraction at the tail ends of cells. This coordinated cytoskeletal reorganization permits cells to move toward a target.³⁵ PI3-kinase and PI (3, 4, 5) P3 have also been widely implicated in controlling cell migration and polarity in a Rac GTPase-dependent manner.³⁵ Members of the Wiskott-Aldrich syndrome protein (WASP) and WASP-family verprolin homologous protein (WAVE) families serve to link Rho GTPases signals to the ARP2/3 complex, leading to actin polymerization that is crucial for the reorganization of the actin cytoskeleton at the leading edge for processes such as cell movement and protrusions.³⁶ Importantly, all three Rho GTPases also regulate microtubule polymerization and assembly of adherens junctions to influence polarity and cell adhesion, respectively.^33,37Likewise, a tensional balance between cell adhesion on the outside and myosin II-based contractility on the inside of the cells is regulated by Rho GTPases.³⁸To explore the role of the Rho GTPases in lens morphogenesis and differentiation, we have targeted the lens Rho GTPases by overexpressing either the C3 exoenzyme (inactivator of RhoA and RhoB) or RhoGDIα (Rho GDP dissociation inhibitor) in a lens-specific manner in transgenic mice and followed their effects developmentally. These two transgenic mouse models exhibited ocular phenotype, including lens opacity (cataract) and microphthalmic eyes. Importantly, various histological, immunofluorescence and biochemical analyses performed in these developing transgenic mice have revealed defective lens morphogenesis, abnormal fiber cell migration, elongation, disrupted cytoskeletal organization and adhesive interactions, along with changes in proteins of the fiber cell gap junctions and water channels.^32,39 These lenses have also shown decreased ERM (ezrin, radixin, moesin) protein phosphorylation,⁴⁰ proteins that are involved in crosslinking of the plasma membrane with actin cytoskeleton,⁴¹ and increased apoptosis.³² Defective fiber cell migration has been found to be more notable in the Rho GDI overexpressing lenses than in the C3 exoenzyme expressing lenses (Fig. 2). The Rho GDI overexpressing lenses have shown a defective membrane localization of Rho, Rac and Cdc42 confirming their inactivation. These data, together with mechanistic studies performed using the lens epithelial cells and the noted effects on cell shape, actin polymerization, myosin phosphorylation and cell adhesive interactions, reveal the importance of Rho GTPase-dependent signaling pathways in processes underlying fiber cell migration, elongation, cytoskeletal and membrane organization and survival in the developing lens.⁷ Lens fiber cell BMC has been found to be localized intensely with Rac GTPase involved in cell migration (our unpublished work). Additionally, the Rho GDI transgenic lenses showed an impaired apical-apical cell-cell interactions between the fiber cells and epithelial cells.³² Moreover, the ruptured posterior capsule and disrupted suture lines in these lenses are indicative of defective BMC organization and activity.³²Open in a separate windowFigure 2Abnormal lens phenotype in the neonatal Rho GDIα overexpressing transgenic mouse. Hematoxylin and eosin-stained sagittal sections of P1 RhoGDIα transgenic eyes reveal abnormal migration and morphology of the posterior lens fibers as compared with the symmetric organization of lens fibers and their migration toward the lens suture in the wild type mouse (reproduced with permission from Maddala et al.)³².Further support for involvement of Rho GTPases in lens fiber cell differentiation and survival has come from studies conducted with chick lens epithelial explants and cultured epithelial cells. Inactivation of Rho kinase or Rac activation by PI3 kinase in chick lens epithelial cells has been reported to induce fiber cell differentiation and survival in association with distinct cortical actin cytoskeletal reorganization, indicating the significance of Rho GTPases in lens fiber cell differentiation and survival.^9,42 Additionally, lens fiber cell elongation and differentiation has been found to be associated with increased myosin light chain (MLC) phosphorylation, and inhibition of MLC phosphorylation regulated by MLC kinase and Rho kinase has induced lens opacity and disruption of cytoskeletal integrity, supporting the importance of myosin II activity in maintaining lens architecture and transparency.¹⁰ Importantly, various growth factors that regulate lens morphogenesis, fiber cell differentiation, and survival have been found to activate Rho and Rac GTPases and to induce MLC phosphorylation, actin cytoskeletal reorganization, and focal adhesion formation in lens epithelial cells.^7,30 In addition to Rho GTPases, inhibition of Src kinase has been shown to induce fiber cell differentiation in association with actin cytoskeletal reorganization and cell adhesive interactions.⁴³ Also, the expression and activation of focal adhesion kinase has been reported to increase in differentiating and migrating lens epithelial cells.⁴⁴ Both these molecules are well recognized to regulate cell migration by participating in the disassembly of cell adhesions at the front of migrating cells.³⁵Additional evidence for the participation of actin cytoskeletal organization and Rho GTPases in lens fiber cell migration and elongation has been derived from the studies of Abi-2 deficient mouse. Abl-interactor adaptor proteins Abi-1 and Abi-2 are linked to the Rac-WAVE-Arp2/3 signaling pathway and regulate actin polymerization and cell-cell adhesive interactions.⁴⁵ Homozygous deletion of Abi-2 in mice has been shown to exhibit ocular phenotype including microphthalmia and lens opacity similar to the Rho GDI overexpressing transgenic mouse eyes noted in previous studies.^23,32 In the absence of Abi-2, the secondary lens fiber orientation, migration and elongation were found to be defective, supporting the importance of Rac-WAVE-Arp2/3 signaling in lens fiber cell migration and cell adhesion.²³ Abi-2 has been shown to localize intensely to the both basal and apical regions of the fiber cells and adherens junctions, and suppression of Abi-2 expression in epithelial cells resulted in impaired adherens junctions and downregulation of actin nucleation promoting factors.²³ The significance of cytoskeletal signaling in lens has also been implicated in Lowe syndrome, a rare X-linked disorder characterized by congenital cataracts, results from mutations in the OCRL1 gene. The OCRL1 protein product (phosphatidylinositol 4, 5 bisphosphate 5-phosphatase) has been shown to participate in Rac GTPase regulated actin cytoskeletal organization, cell migration, and cell adhesion in various cell types.⁴⁶ Finally, Wnt/PCP signaling via activation of Rho GTPases has been suggested to control lens morphogenesis, fiber cell migration and differentiation.²⁶Importantly, given how the activity of the Rho GTPases is regulated by external cues and various effector proteins, a detailed understanding of the regulation of Rho GTPase signaling is necessary for a better appreciation of their role in lens morphogenesis, fiber cell elongation and differentiation, and tensional homeostasis. Further mechanistic studies are critical to unravel the specific role(s) of Rho GTPases and other cytoskeletal regulatory mechanisms involved in regulating the formation and disassembly of fiber cell basal and apical membrane complexes, fiber cell lateral membrane remodeling, and fiber cell-cell adhesive interactions during lens differentiation. Very little is known in terms of the assembly of different cell adhesive molecules at the apical-apical interface between the lens fibers and epithelial cells. We are only beginning to glimpse the regulatory networks involved in the regulation of fiber cell elongation, polarity, migration and adhesion. Many challenging questions remain: for example, how are the pathways regulating migration, basal and apical membrane complexes, and tensional homeostasis controlled by extracellular signals, and how are they integrated during fiber cell migration, suture formation, and packing? Novel insights into the molecular mechanisms regulating these cellular processes are expected to advance our understanding of lens morphogenesis, function and cataractogenesis.     

Chromosome segregation during female meiosis in C. elegans: A tale of pushing and pulling

The role of the kinetochore during meiotic chromosome segregation in C. elegans oocytes has been a matter of controversy. Danlasky et al. (2020. J. Cell. Biol. https://doi.org/10.1083/jcb.202005179) show that kinetochore proteins KNL-1 and KNL-3 are required for early stages of anaphase during female meiosis, suggesting a new kinetochore-based model of chromosome segregation.

Meiosis consists of two consecutive chromosome segregation events preceded by a single round of DNA replication. Homologous chromosomes are separated in meiosis I, which is followed by sister chromatid separation in meiosis II to produce haploid gametes. Both of these stages require chromosomes/chromatids to align during metaphase before separating to opposite poles during anaphase. During mitosis, microtubules emanating from centrosomes at opposite poles of the cell bind chromosomes through a multiprotein complex called the kinetochore, allowing chromosomes to be pulled apart (1, 2). This segregation event takes place in two stages: anaphase A, where chromosomes are pulled toward spindle poles due to microtubule depolymerization, and anaphase B, where spindle poles themselves move farther apart, taking the attached chromosomes with them (3, 4). In many organisms, including mammals, oocytes lack centrosomes, and it has been of great interest to clarify the mechanisms used to ensure chromosomes are properly segregated during female meiosis (5, 6). Caenorhabditis elegans has served as a model for studying both mitosis and meiosis, but the mechanisms operating during female meiosis have been a matter of debate and controversy.In 2010, Dumont et al. showed that the kinetochore is required for chromosome alignment and congression during metaphase (7). However, they suggested that chromosome segregation was the result of microtubule polymerization between the segregating chromosomes (Fig. 1), resulting in a pushing force exerted onto chromosomes toward the spindle poles in a largely kinetochore-independent manner (7). This mechanism was also supported by the finding that CLIP-associated protein (CLASP)–dependent microtubule polymerization between the segregating chromosomes is essential for chromosome separation (8). An alternative model suggested that chromosomes are transported through microtubule-free channels toward the spindle poles by the action of dynein (9). Later evidence put in doubt a role for dynein and favored a model in which chromosomes initially separate when the spindle shortens and the poles overlap with chromosomes in an anaphase A–like mechanism. This is then followed by separation of chromosome-bound poles by outward microtubule sliding in an anaphase B–like fashion (10). However, because microtubules emanating from the spindle poles are not required to separate the homologous chromosomes but microtubules between the separating chromosomes are (8), this model is unlikely, at least as an explanation for mid-/late-anaphase movement. Furthermore, although lateral microtubule interactions with chromosomes predominate during metaphase of C. elegans oocyte meiosis, cryo-electron tomography data described end-on attachments between the separating chromosomes as anaphase progresses (11). This led to the suggestion that lateral microtubule interactions with chromosomes are responsible for the initial separation, but microtubule polymerization between the separating chromosomes is required for the later stages of segregation (11). The mechanisms involved in this initial separation have remained obscure. In this issue, Danlasky et al. show that the kinetochore is in fact required for the initial stages of chromosome segregation during female meiosis—an important step forward in our understanding of the mechanisms governing acentrosomal chromosome segregation (12).Open in a separate windowFigure 1.Some of the key findings in Danlasky et al. Kinetochore proteins surround the outer surface of the chromosomes, resulting in a characteristic cup shape. As anaphase progresses, chromosomes come into close contact to the spindle poles (anaphase A). Chromosome stretching is provided by KNL-1, MIS-12 (KNL-3), and NDC-80 (KMN)–dependent forces. Once the spindle starts elongating (anaphase B), central spindle microtubules provide the pushing forces for chromosome segregation. At this stage, kinetochore proteins also occupy the inward face of separating chromosomes. Upon KMN network depletion, bivalents flatten, and chromosome congression and alignment are defective. Anaphase A chromosome movement is almost absent, which leads to error-prone anaphase B.By simultaneously depleting kinetochore proteins KNL-1 and KNL-3 in C. elegans, Danlasky et al. observed the meiotic chromosome congression and alignment defects described in previous studies (7). However, this double-depletion phenotype displayed three key characteristics that suggested a role for kinetochores in chromosome segregation, which are discussed below.The kinetochore is required for bivalent stretching. It was previously shown that the bivalent chromosomes stretch before the initiation of segregation (10). Danlasky et. al found that this stretching of the chromosomes did not occur when KNL-1,3 were depleted, indicating that the kinetochore is required for this process (Fig. 1). Together with the observation that kinetochore proteins appear to extend toward the spindle poles, this finding suggested that pulling forces resulting from the interaction between the kinetochore and spindle microtubules are occurring during metaphase/preanaphase (Fig. 1).The kinetochore is required for anaphase A. In C. elegans female meiosis, anaphase A occurs when homologous chromosomes begin to separate during spindle shortening, and anaphase B when the chromosomes separate alongside the spindle poles (10). Danlasky et al. observed that KNL-1,3 depletion drastically reduced the velocity of anaphase A, as chromosomes only separated when spindle poles began to move apart. This indicated that pulling forces caused by the interaction between the kinetochore and spindle microtubules are also important for the initial separation of homologous chromosomes in anaphase A.The kinetochore is required for proper separation of homologous chromosomes. In KNL-1,3 depletion strains, 60% of bivalents failed to separate before segregation began, resulting in intact bivalents being pulled to the same spindle pole (Fig. 1). This failure of homologous chromosomes to separate was not thought to be a result of KNL-1,3 depletion interfering with the cleavage of cohesin that holds the two homologous chromosomes together because (a) separase and AIR-2^AuroraB, both of which are required for cohesin cleavage, localized normally during metaphase and anaphase, and (b) bivalents separated by metaphase II. This leaves the possibility open that the failure of bivalents to separate was due to the disrupted pulling forces thought to be important in bivalent stretching and anaphase A.Altogether, these data strongly indicate that the kinetochore is required not only for chromosome congression and alignment but also for the early stages of homologue separation. Anaphase B occurred successfully in the absence of KNL-1,3 but was more error prone, likely as a result of the earlier congression and anaphase A defects. While it is clear that chromosome masses do segregate in the absence of the kinetochore, this segregation is highly erroneous as a result of defects during the earlier stages of segregation in anaphase A (Fig. 1).The findings of Danlasky et al. raise testable hypotheses that could significantly enhance our understanding of acentrosomal chromosome segregation. Further investigation of the proposed pulling forces required during metaphase and early anaphase will be of great interest. Additionally, a more detailed analysis of the dynamic localization of separase and Securin, as well as assessing successful cohesin cleavage when KNL-1,3 are depleted, would back up the assertion that the failure of homologous chromosomes to separate was not due to the kinetochore impacting cohesin cleavage. It has previously been shown that the CLASP orthologue CLS-2 in C. elegans localizes to the kinetochore surrounding the bivalent chromosomes during metaphase before relocalizing to the central spindle during anaphase (7, 8, 13). It will be interesting to examine whether this key microtubule-stabilizing protein contributes to anaphase A pulling forces alongside its essential role in microtubule polymerization between chromosomes in anaphase B (8).While the regulation of proper chromosome segregation during acentrosomal meiosis in C. elegans is not yet fully understood, Danlasky et al.’s results represent a significant step forward in this endeavor by showing that the kinetochore is in fact required for the early stages of chromosome segregation.     

Shoulder muscle activity during pushing, pulling, elevation and overhead throw.

PURPOSE: The aim of this study was to compare the muscle activity of recreational athletes and professional javelin throwers during pull, push, and elevation of upper extremities and during overhead throw. SCOPE: Nine professional javelin throwers and 16 recreational athletes without shoulder problems were studied. Signals were recorded by surface EMG from eight different muscles. The results obtained from the muscles of upper extremities of throwers were compared with those of recreational athletes. CONCLUSION: The different neuromuscular control of professional throwers caused a more profitable muscle activity. Differences during the overhead throw were more significant. The deltoid muscle and rotator cuff of recreational athletes showed stronger activity than those of throwers during pull, push and elevation. The deltoid muscle and the rotator cuff of professional throwers showed stronger activity during overhead throw. Studying the detailed characteristics of muscle activity pattern (differences in length of activity periods, MVC% of muscles and time broadness among peak muscle activities in percent of total time of a movement cycle) may provide a basis for better understanding improved performance and help in planning proper rehabilitation protocol.     

Hair pulling: a review

Hair pulling has been reported in humans, six different non-human primate species, mice, guineapigs, rabbits, sheep and muskox, dogs and cats. This behaviour seems to occur only in subjects who are confined in an artificial environment. It has been classified as a mental disorder in humans, as a behavioural pathology in animals. The hair is not only pulled but also, in most species, ingested. Hair pulling can be both self-directed and partner-directed, contains elements of aggression, manifests more often in females than in males, is associated with psychogenic distress, and resists treatment. Research data collected from affected animals are probably not normative, hence scientifically unreliable. The preemptive correction of husbandry deficiencies causing long-term stress may prevent the development of this bizarre behaviour in healthy subjects.     

Pushing, pulling and trapping--modes of motor protein supported protein translocation

Protein translocation across the cellular membranes is an ubiquitous and crucial activity of cells. This process is mediated by translocases that consist of a protein conducting channel and an associated motor protein. Motor proteins interact with protein substrates and utilize the free energy of ATP binding and hydrolysis for protein unfolding, translocation and unbinding. Since motor proteins are found either at the cis- or trans-side of the membrane, different mechanisms for translocation have been proposed. In the Power stroke model, cis-acting motors are thought to push, while trans-motors pull on the substrate protein during translocation. In the Brownian ratchet model, translocation occurs by diffusion of the unfolded polypeptide through the translocation pore while directionality is achieved by trapping and refolding. Recent insights in the structure and function of the molecular motors suggest that different mechanisms can be employed simultaneously.     

Metabolic effects of training in humans: a 31P-MRS study

The purpose of this study was to determine the feasibility of measuring with 31P nuclear magnetic resonance the effects of an endurance training program on the high-energy phosphate metabolism of exercising human skeletal muscle. The system used included a 1.9-T 30-cm-bore Oxford Systems superconducting magnet, a PhosphoEnergetics spectrometer, and a modified Cybex isokinetic ergometer. Seven healthy human volunteers exercised their wrist flexor muscles 20 min/day 5 days/wk for 8 wk. Testing before and after the training period consisted of a performance test to measure muscle functional capacity and a ramp test to measure the work-energy cost relationship of the exercising muscles. The results indicate that the subjects had a significant increase in their work output on the 10-min performance test after training. They also exhibited an increase in the work-energy cost relationship on the ramp test as indicated by a decrease in peak Pi-to-phosphocreatine ratio and an increase in pH at the same relative power output after training. These results indicate that 1) the training program was sufficient to elicit a training effect and 2) this effect was observed with 31P nuclear magnetic resonance as an increased potential for oxidative metabolism, particularly at the high exercise levels.     

Comparison of training and game demands of national level cricketers

Although the physiological demands of cricket match play are emerging, the demands of contemporary training practices have not been reported. The aim of this study was to quantify the physiological demands of selected cricket training activities and compare these to known match demands. Twenty-eight different training activities were monitored in national academy level cricketers (n = 42) using global positioning system units during a 14-week residential training program. The training activities were classified into 3 categories: conditioning sessions (n = 8), skill sessions (n = 9), and game simulations (n = 11). Conditioning sessions were further classified into high- (n = 4) and low- (n = 4) intensity drills. Time-motion measures included movement patterns (walk, jog, run, stride, and sprint distances), total distance covered, number of sprints, number of high-intensity efforts, maximum speed, and recovery time between high-intensity efforts. Inferential statistics were used to quantify magnitudes of difference between various training drills. Movement patterns were then compared to recently published game data (Twenty20, One-Day, and Multiday games) from the same sample group of cricketers. Conditioning drills were twice as long in duration as skill drills and twice as intense as both the skill and game simulation drills. Exercising heart rates were 9-26% and lactate levels up to 3.5 times higher in conditioning compared to other training drills. Conditioning drills matched or exceeded (up to 10 b·min?1; 5%) peak game heart rates. Conversely, skill and simulation drills replicated mean game heart rates for some, but not all positions. In conclusion, training replicates or exceeds cricket match demands in conditioning-type drills but not in simulation or skill-based drills. Modification of skill and simulation training drill practices will ensure closer replication of match and training demands.     

Metabolic and cardiovascular adjustment to arm training

Maximal and submaximal metabolic and cardiovascular measures and work capacity were studied in control (n = 7) and experimental (n = 9) subjects (S's) during arm work prior to and following 10 wk of interval arm training. These measures were oxygen uptake (VO2), minute ventilation (VE), heart rate (HR), respiratory exchange ratio (R), cardiac output (Q), stroke volume (SV), and arteriovenous oxygen difference ((a--v)O2 diff). In addition, maximal oxygen uptake (VO2max) was measured in both groups during treadmill running. Experimental S's showed significant increases (P less than 0.01) in peak VO2 (438 ml.min-1), max VE (17.7 l.min-1), max (a--v)O2 diff (20.8 ml.l-1), and work time (9.2 min) during arm ergometry, while maximum values of Q, SV, HR, and R remained unchanged. In addition, submaximal heart rates were significantly lower during arm ergometry after training. VO2max during treadmill running remained essentially unchanged. No changes in metabolic and physiological measures were noted for the controls after the 10-wk training period. The results support the concept of training specificity for VO2max, and indicate that the improvement in peak VO2 in arm ergometry reflects enhanced oxygen utilization due to an expanded (a--v)O2 diff.     

Peripheral circulatory responses in vivo from regional brachial biceps and lumbar muscles in healthy men and women during pushing and pulling exercise

Background: Although women have been performing increasingly more manual labor in the workplace in the past 2 decades, their physiological responses and gender-based differences in muscle microvascularity during occupational activities have not yet been extensively documented.Objective: This study assessed gender differences and tissue heterogeneity in peripheral circulatory responses from 2 muscle groups during pushing and pulling exercise until volitional exhaustion. Methods: In healthy men and women, near-infrared spectroscopy was used to determine peripheral responses, oxygenation, and blood volume simultaneously from the right biceps brachii and lumbar erector spinae. Pulmonary oxygen uptake was assessed using a metabolic measurement cart.Results: Although the 11 men who participated in the study demonstrated greater pulmonary oxygen uptake and power output at volitional exhaustion, their peak peripheral responses for both muscles were similar to those of the 11 women participating. In both sexes, oxygenations trends decreased in both muscles with an increase in workload. However, whereas blood volume increased in the biceps, it decreased in the lumbar muscle in both sexes. At 20% to 60% levels of peak pulmonary oxygen uptake, the percent change in peripheral bicep responses was greater for men than for women (P < 0.05). In contrast, women demonstrated greater change in lumbar muscle oxygenation compared with men at 40% to 60% of peak pulmonary oxygen uptake (P < 0.05).Conclusions: Similar peripheral responses for biceps and lumbar muscles at the point of volitional exhaustion suggest that gender differences in pulmonary oxygen uptake are independent of oxygen extraction or delivery across the muscle groups monitored. However, at submaximal levels of exercise, the peripheral changes in each muscle were gender dependent. Although biceps and lumbar muscles are 2 discrete muscle groups, based on the heterogeneity found in the blood volume trends it is likely that oxygen supply and demand are regulated by muscle location and muscle fiber characteristics. Overall, gender-based assessment of occupational activities should incorporate both pulmonary and peripheral circulatory responses to understand each sex's performance effectiveness.