Direct measurement of the lamellipodial protrusive force in a migrating cell

There has been a great deal of interest in the mechanism of lamellipodial protrusion (Pollard, T., and G. Borisy. 2003. Cell. 112:453–465). However, one of this mechanism's endpoints, the force of protrusion, has never been directly measured. We place an atomic force microscopy cantilever in the path of a migrating keratocyte. The deflection of the cantilever, which occurs over a period of ∼10 s, provides a direct measure of the force exerted by the lamellipodial leading edge. Stall forces are consistent with ∼100 polymerizing actin filaments per micrometer of the leading edge, each working as an elastic Brownian ratchet and generating a force of several piconewtons. However, the force-velocity curves obtained from this measurement, in which velocity drops sharply under very small loads, is not sensitive to low loading forces, and finally stalls rapidly at large loads, are not consistent with current theoretical models for the actin polymerization force. Rather, the curves indicate that the protrusive force generation is a complex multiphase process involving actin and adhesion dynamics

Cognitive Dysfunction Is Sustained after Rescue Therapy in Experimental Cerebral Malaria, and Is Reduced by Additive Antioxidant Therapy

Neurological impairments are frequently detected in children surviving cerebral malaria (CM), the most severe neurological complication of infection with Plasmodium falciparum. The pathophysiology and therapy of long lasting cognitive deficits in malaria patients after treatment of the parasitic disease is a critical area of investigation. In the present study we used several models of experimental malaria with differential features to investigate persistent cognitive damage after rescue treatment. Infection of C57BL/6 and Swiss (SW) mice with Plasmodium berghei ANKA (PbA) or a lethal strain of Plasmodium yoelii XL (PyXL), respectively, resulted in documented CM and sustained persistent cognitive damage detected by a battery of behavioral tests after cure of the acute parasitic disease with chloroquine therapy. Strikingly, cognitive impairment was still present 30 days after the initial infection. In contrast, BALB/c mice infected with PbA, C57BL6 infected with Plasmodium chabaudi chabaudi and SW infected with non lethal Plasmodium yoelii NXL (PyNXL) did not develop signs of CM, were cured of the acute parasitic infection by chloroquine, and showed no persistent cognitive impairment. Reactive oxygen species have been reported to mediate neurological injury in CM. Increased production of malondialdehyde (MDA) and conjugated dienes was detected in the brains of PbA-infected C57BL/6 mice with CM, indicating high oxidative stress. Treatment of PbA-infected C57BL/6 mice with additive antioxidants together with chloroquine at the first signs of CM prevented the development of persistent cognitive damage. These studies provide new insights into the natural history of cognitive dysfunction after rescue therapy for CM that may have clinical relevance, and may also be relevant to cerebral sequelae of sepsis and other disorders

Cell motility: the integrating role of the plasma membrane

The plasma membrane is of central importance in the motility process. It defines the boundary separating the intracellular and extracellular environments, and mediates the interactions between a motile cell and its environment. Furthermore, the membrane serves as a dynamic platform for localization of various components which actively participate in all aspects of the motility process, including force generation, adhesion, signaling, and regulation. Membrane transport between internal membranes and the plasma membrane, and in particular polarized membrane transport, facilitates continuous reorganization of the plasma membrane and is thought to be involved in maintaining polarity and recycling of essential components in some motile cell types. Beyond its biochemical composition, the mechanical characteristics of the plasma membrane and, in particular, membrane tension are of central importance in cell motility; membrane tension affects the rates of all the processes which involve membrane deformation including edge extension, endocytosis, and exocytosis. Most importantly, the mechanical characteristics of the membrane and its biochemical composition are tightly intertwined; membrane tension and local curvature are largely determined by the biochemical composition of the membrane and the biochemical reactions taking place; at the same time, curvature and tension affect the localization of components and reaction rates. This review focuses on this dynamic interplay and the feedbacks between the biochemical and biophysical characteristics of the membrane and their effects on cell movement. New insight on these will be crucial for understanding the motility process

Iron Behaving Badly: Inappropriate Iron Chelation as a Major Contributor to the Aetiology of Vascular and Other Progressive Inflammatory and Degenerative Diseases

The production of peroxide and superoxide is an inevitable consequence of aerobic metabolism, and while these particular "reactive oxygen species" (ROSs) can exhibit a number of biological effects, they are not of themselves excessively reactive and thus they are not especially damaging at physiological concentrations. However, their reactions with poorly liganded iron species can lead to the catalytic production of the very reactive and dangerous hydroxyl radical, which is exceptionally damaging, and a major cause of chronic inflammation. We review the considerable and wide-ranging evidence for the involvement of this combination of (su)peroxide and poorly liganded iron in a large number of physiological and indeed pathological processes and inflammatory disorders, especially those involving the progressive degradation of cellular and organismal performance. These diseases share a great many similarities and thus might be considered to have a common cause (i.e. iron-catalysed free radical and especially hydroxyl radical generation). The studies reviewed include those focused on a series of cardiovascular, metabolic and neurological diseases, where iron can be found at the sites of plaques and lesions, as well as studies showing the significance of iron to aging and longevity. The effective chelation of iron by natural or synthetic ligands is thus of major physiological (and potentially therapeutic) importance. As systems properties, we need to recognise that physiological observables have multiple molecular causes, and studying them in isolation leads to inconsistent patterns of apparent causality when it is the simultaneous combination of multiple factors that is responsible. This explains, for instance, the decidedly mixed effects of antioxidants that have been observed, etc...Comment: 159 pages, including 9 Figs and 2184 reference

Direct measurement of protrusional forces of migrating cells

Cellular migration is a fundamental process in nature. It is very important, for instance, in wound healing and metastasis. Therefore the movement of cells is in the focus of scientific research for some time and many of the molecular details have been discovered in recent years. A still open question is the actual mechanism generating the protrusion force at the leading edge of a cell. One reason is the lack of direct data measuring force and velocity of the leading edge. I have measured, for the first time, the protrusion force of a well defined local area of the lamellipodium. In this work, an AFM (�Atomic Force Microscope�) � cantilever was oriented perpendicular to the substrate and used as a flexible obstacle, which is hit by the cell. The deflection of the cantilever, which is proportional to the force exerted by the cantilever and the cell, can be determined by video-microscopy.A completely new set-up was constructed to host the cantilever and to position it only 100nm above the glass-substrate with sufficient stability, the possibility to move the sample laterally and vertically by piezo control, and to monitor the sample by transillumination optical microscopy.A protocol was established to cultivate keratocyte cells used in this study. Keratocytes are a primary epithelial cell-line from rainbow trout, which show fast movement at nearly constant velocity.For control measurements fluorescence microscopy, atomic force microscopy and reflection-interference-contrast-microscopy were used.Maximum measured protrusion forces were in the range of 1.2nN for the lamellipodium, measured traction forces for whole cells were around 40nN. From the deflection data of the cantilever force-velocity diagrams could be generated which allows us to compare our results with theoretical model describing lamellar protrusion. The data do not coincide with the elastic brownian ratchet, which has been designed for another system. There is a good agreement with the end-motor model, which proposes the existence of an active motor molecule at the end of the actin filaments, which has not been discovered yet

Direkte Messung der Protrusionskraft wandernder Zellen

Cellular migration is a fundamental process in nature. It is very important, for instance, in wound healing and metastasis. Therefore the movement of cells is in the focus of scientific research for some time and many of the molecular details have been discovered in recent years. A still open question is the actual mechanism generating the protrusion force at the leading edge of a cell. One reason is the lack of direct data measuring force and velocity of the leading edge. I have measured, for the first time, the protrusion force of a well defined local area of the lamellipodium. In this work, an AFM (�Atomic Force Microscope�) � cantilever was oriented perpendicular to the substrate and used as a flexible obstacle, which is hit by the cell. The deflection of the cantilever, which is proportional to the force exerted by the cantilever and the cell, can be determined by video-microscopy.A completely new set-up was constructed to host the cantilever and to position it only 100nm above the glass-substrate with sufficient stability, the possibility to move the sample laterally and vertically by piezo control, and to monitor the sample by transillumination optical microscopy.A protocol was established to cultivate keratocyte cells used in this study. Keratocytes are a primary epithelial cell-line from rainbow trout, which show fast movement at nearly constant velocity.For control measurements fluorescence microscopy, atomic force microscopy and reflection-interference-contrast-microscopy were used.Maximum measured protrusion forces were in the range of 1.2nN for the lamellipodium, measured traction forces for whole cells were around 40nN. From the deflection data of the cantilever force-velocity diagrams could be generated which allows us to compare our results with theoretical model describing lamellar protrusion. The data do not coincide with the elastic brownian ratchet, which has been designed for another system. There is a good agreement with the end-motor model, which proposes the existence of an active motor molecule at the end of the actin filaments, which has not been discovered yet

Dehydration stability of amyloid fibrils studied by AFM

Atomic force microscopy was used to investigate the stability of dehydrated amyloid fibrils formed by human islet polypeptide (IAPP) and A beta(1-42) peptides. IAPP amyloid fibrils were imaged in liquid (hydrated state) and in air (dehydrated). In addition, fibrils dried on the mica surface were rehydrated and re-examined both in liquid and in air (after consecutive redrying). As reported previously, the initial drying process does not result in any major change in the amyloid appearance and the dimensions of the fibrils are preserved. However, when once-dried samples are rehydrated, fibril stability is lost. The fibrils disintegrate into small particles that are attached to the mica surface. This process is further confirmed by studies of the rehydrated samples after drying, on which the morphology of the fibrils is clearly changed. Similar behavior is observed for A beta(1-42) amyloid fibrils, which are apparently stable on first drying, but disintegrate on rehydration. The observed change indicates that dehydration is causing a change in the internal structure of the amyloid fibrils. This has important implications for studies of amyloid fibrils by other techniques. Due to the potential influence of hydration and sample history on amyloid structure, preparation and study of amyloid samples with controlled humidity requires more consideration

Montagem, testes operacionais e validação de uma unidade laboratorial para extração de compostos de matrizes vegetais utilizando fluidos pressurizados ou supercríticos como solventes

Extractions of compounds still use traditional methods, leaving solvent residues in the extracts. An alternative is the use of high-pressure solvents such as supercritical CO2 and pressurized liquefied petroleum gas (LPG). Based on this context, the objective of this work was to assemble an equipment for high-pressure extractions using CO2 or pressurized LPG as solvent. The project and assembling were divided into 3 steps. In the first step, the equipment was designed. Also, aluminum bars, pipes, valves, pressure gauges, thermostatic baths, a pump for solvent pressurization, a digital temperature controller, a band heater and a reactor with filters were purchased. In the second step, some adaptations were done in the structure and its components for improving the operation. In the third step, the equipment was effectively assembled. Thereafter, operational validation tests were performed in order to verify the operation and the necessity of adjustments. In addition, a Standard Operating Procedure was created in order to standardize the next operations. The equipment is suitable for the extraction of bioactive compounds from different plant matrices, providing satisfactory extraction yields.As extrações de compostos de matrizes vegetais ainda utilizam métodos tradicionais, deixando resíduos dos solventes nos extratos. Uma alternativa é a utilização de solventes a alta pressão, como CO2 no estado supercrítico e gás liquefeito de petróleo (GLP) pressurizado. Como objetivo deste trabalho, foi proposta a montagem de uma unidade para extração utilizando CO2 supercrítico ou GLP pressurizado como solvente. O projeto e a montagem foram divididos em 3 etapas. Na primeira etapa, foi realizado o desenho e foram adquiridas as ferramentas, barras de alumínio para montagem da estrutura, tubulações, válvulas, manômetros, banhos termostatizados, uma bomba para pressurização do solvente, um controlador digital de temperatura, uma manta de aquecimento reatores com filtros. Na segunda etapa, foram realizadas adaptações na estrutura e seus componentes para fins de melhorias. Na terceira etapa, foi realizada a montagem dos componentes. Após a montagem, foram realizados testes operacionais de validação com o objetivo de verificar o funcionamento da unidade e a necessidade de eventuais ajustes. Além disso, foi criado um Procedimento Operacional Padrão para as operações seguintes se manterem padronizadas. Os resultados demonstraram que a unidade é adequada para a extração de compostos bioativos de diferentes matrizes vegetais, fornecendo rendimentos satisfatórios de extrato