Mechanically induced electrical potentials of articular cartilage

While there is increasing evidence that chondrocytes are affected by mechanically induced stimuli, endogenous force-related electrical potentials within articular cartilage have been so far observed only in-vitro. Using a porcine ex-vivo model (German Land Race), 8 knee joints were explanted and exposed to mechanical force (up to 800 N) using a special device. Electrodes were inserted into the cartilage matrix. With an amplifier and an A/D transducer the changes of electrical voltage between the electrodes as well as those of the force were recorded online and simultaneously on a computer. Additionally, we located one pair of electrodes on the surface of the cartilage tissue to detect electrical fields outside the cartilage tissue. In relation to the applied force we observed that electrical potentials derived from inside and outside the articular cartilage showed a correspondence. When an alternating force with an amplitude of 360 N and a frequency of about 0.2 Hz was periodically applied, we measured peak amplitudes ranging from 2.1 to 5.5 mV within the cartilage tissue with electrical negativity within the weight bearing area of the cartilage tissue. The measured voltages depended on the applied force, the location of the electrodes, and on anatomical variations. We found an almost linear relation between the magnitude of the applied force and the recorded voltage. With the help of the electrodes located outside and within the cartilage tissue, we were able to show that force dependent fields are generated inside the cartilage. There are several theories explaining the origin of these electrical phenomena, many of them focusing on the negative charges of the proteoglycans in relation to the flow of interstitial fluid and ions under compression. However, the consequences of these phenomena are yet not clear.     

Deformations in actin comets from rocketing beads

The mechanical and dynamical properties of the actin network are essential for many cellular processes like motility or division, and there is a growing body of evidence that they are also important for adhesion and trafficking. The leading edge of migrating cells is pushed out by the polymerization of actin networks, a process orchestrated by cross-linkers and other actin-binding proteins. In vitro physical characterizations show that these same proteins control the elastic properties of actin gels. Here we use a biomimetic system of Listeria monocytogenes, beads coated with an activator of actin polymerization, to assess the role of various actin-binding proteins in propulsion. We find that the properties of actin-based movement are clearly affected by the presence of cross-linkers. By monitoring the evolution of marked parts of the comet, we provide direct experimental evidence that the actin gel continuously undergoes deformations during the growth of the comet. Depending on the protein composition in the motility medium, deformations arise from either gel elasticity or monomer diffusion through the actin comet. Our findings demonstrate that actin-based movement is governed by the mechanical properties of the actin network, which are fine-tuned by proteins involved in actin dynamics and assembly.     

Mechanically induced helix-coil transition in biopolymer networks

The quasi-equilibrium evolution of the helical fraction occurring in a biopolymer network (gelatin gel) under an applied stress has been investigated by observing modulation in its optical activity. Its variation with the imposed chain extension is distinctly nonmonotonic and corresponds to the transition of initially coiled strands to induced left-handed helices. The experimental results are in qualitative agreement with theoretical predictions of helices induced on chain extension. This new effect of mechanically stimulated helix-coil transition has been studied further as a function of the elastic properties of the polymer network: crosslink density and network aging.     

Mechanically induced reflex responses in human triceps brachii

The short and long latency reflex responses of human triceps brachii muscle were recorded in 14 healthy volunteers. An electromechanical hammer was used to stretch the muscle and recordings were made from a surface electromyogram. The monosynaptic tendon reflex occurred at a mean latency of 12.5 ms (SE 0.7 ms). Later responses were observed in activated conditions (weak force production, preparatory period) at a mean latency of 62.8 ms (SE 3.5 ms). The amplitude of the short latency reflex increased during weak tension, the long latency reflex amplitude seemed to increase during the preparatory period testing. The amplitude increases can be attributed to increased lower motoneuron excitability even during weak voluntary activity. The tendency towards an increased amplitude during the preparatory period may be connected with the higher regulation of the long latency reflex.     

Mechanically induced orientation of adult rat cardiac myocytes in vitro

Summary A population of freshly isolated adult rat cardiac myocytes is spatially oriented using a computerized mechanical cell stimulator device for tissue cultured cells. A continuous unidirectional stretch of the substratum at 60 to 400 μm/min for 120 to 30 min, respectively, during the cell attachment period in serum-free medium induces a significant three-fold increase in the number of rod-shaped myocytes oriented parallel to the direction of movement. The myocytes orient less well with unidirectional substratum stretching after their adhesion to the substratum. In contrast, adult myocytes plated onto a substratum undergoing continuous 10% stretch-relaxation cycling show no significant change in myocyte orientation or cytoskeletal organization. Orientation of rod-shaped myocytes is dependent on several factors other than the type of mechanical activity. These include: a) the speed of substratum movement; b) the final stretch amplitude; and c) the timing between initiation of substratum stretching and adhesion of myocytes to the substratum. Oriented adult rod shaped myocytes representing 65 to 70% of the total myocyte population in this model system can now be submitted to different patterns of repetitive mechanical stimulation for the study of stretch-induced alterations in cell growth and gene expression. This work was supported by grants AR36266, AR39998, and RR05818 from the National Institutes of Health, Bethesda, MD, and grant NAG2-414 from the National Aeronautics and Space Administration, Washington, DC. J.-L. Samuel was a recipient from the Foundation pour la Recherche Médicale.     

Mechanically induced avoidance response of chloroplasts in fern protonemal cells.

Cell response to mechanical stimulation was investigated at a subcellular level in protonemal cells of the fern Adiantum capillus-veneris L. by pressing a small part of the cell with a microcapillary. In cells receiving local stimulation, the chloroplasts moved away from the site of stimulation, whereas the nuclei failed to show such avoidance movement. Mechanical stimulation for a period as short as 0.3 min was enough to induce the avoidance response to a maximal level. The avoidance movement of chloroplasts started within 30 min and the plateau level of avoidance was attained around 2 h after stimulation. By tracing the movement of chloroplasts during the response, it was shown that the mobility of chloroplasts near the stimulation site increased transiently within 1 h after the stimulation. After 2 to 3 h, it slowed down to the control level without stimulation. The avoidance response was inhibited by 0.1 mM cytochalasin B and 25 mM 2, 3-butanedione monoxime but not by 3.3 microM amiprophosmethyl or 5 mM colchicine. These findings indicate that the protonemal cells were very sensitive to mechanical stimulation and that chloroplasts moved away from the mechanically stimulated site through the actomyosin motile system.     

Annexin 2 has an essential role in actin-based macropinocytic rocketing

Annexin 2 is a Ca(2+) binding protein that binds to and aggregates secretory vesicles at physiological Ca(2+) levels [1] and that also associates Ca(2+) independently with early endosomes [2, 3]. These properties suggest roles in both exocytosis and endocytosis, but little is known of the dynamics of Annexin 2 distribution in live cells during these processes. We have used evanescent field microscopy to image Annexin 2-GFP in live, secreting rat basophilic leukemia cells and in cells performing pinocytosis. Although we found no evidence of Annexin 2 involvement in exocytosis, we observed an enrichment of Annexin 2-GFP in actin tails propeling macropinosomes. The association of Annexin 2-GFP with rocketing macropinosomes was specific because Annexin 2-GFP was absent from the actin tails of rocketing Listeria. This finding suggests that the association of Annexin 2 with macropinocytic rockets requires native pinosomal membrane. Annexin 2 is necessary for the formation of macropinocytic rockets since overexpression of a dominant-negative Annexin 2 construct abolished the formation of these structures. The same construct did not prevent the movement of Listeria in infected cells. These results show that recruitment of Annexin 2 to nascent macropinosome membranes 16656is an essential prerequisite for actin polymerization-dependent vesicle locomotion.     

Actin filament uncapping localizes to ruffling lamellae and rocketing vesicles

Regulated actin filament assembly is critical for eukaryotic cell physiology. Actin filaments are polar structures, and those with free high affinity or barbed ends are crucial for actin dynamics and cell motility. Actin filament barbed-end-capping proteins inhibit filament elongation after binding, and their regulated disassociation is proposed to provide a source of free filament ends to drive processes dependent on actin polymerization. To examine whether dissociation of actin filament capping proteins occurs with the correct spatio-temporal constraints to contribute to regulated actin assembly in live cells, I measured the dissociation of an actin capping protein, gelsolin, from actin in cells using a variation of fluorescence resonance energy transfer (FRET). Uncapping was found to occur in cells at sites of active actin assembly, including protruding lamellae and rocketing vesicles, with the correct spatio-temporal properties to provide sites of actin filament polymerization during protrusion. These observations are consistent with models where uncapping of existing filaments provides sites of actin filament elongation.     

Kinetics of phosphatidylinositol-3-phosphate acquisition differ between IgG bead-containing phagosomes and Mycobacterium tuberculosis-containing phagosomes

A key aspect of Mycobacterium tuberculosis pathogenesis is the ability of the bacteria to survive within the host macrophage. A phagosome containing an IgG-coated bead matures into a lysosomal compartment as evidenced by a decrease in pH and an increased acquisition of hydrolytic enzymes. In contrast, when M. tuberculosis is phagocytosed, the maturation of the bacteria-containing phagosome is arrested, and the bacterium resides within a vacuole that retains characteristics of early endosomal compartments. M. tuberculosis-containing phagosomes are delayed in the recruitment of the early endosome autoantigen EEA1. Acquisition of EEA1 is dependent on the presence of phosphatidylinositol-3-phosphate (PI-3-P) generated by the kinase Vps34. We tested the hypothesis that delayed recruitment of EEA1 was due to altered kinetics of PI-3-P accumulation at the phagosomal membrane. Biochemical analysis of the phosphatidylinositol phosphates on M. tuberculosis-containing phagosomes revealed that PI-3-P acquisition was markedly retarded and reduced in comparison to IgG bead-containing phagosomes. Given the role these lipids play in the regulation of phagosome maturation these findings have implications with respect to the mechanisms behind the arrest of phagosome maturation.     

Mechanically induced titin kinase activation studied by force-probe molecular dynamics simulations

The conversion of mechanical stress into a biochemical signal in a muscle cell requires a force sensor. Titin kinase, the catalytic domain of the elastic muscle protein titin, has been suggested as a candidate. Its activation requires major conformational changes resulting in the exposure of its active site. Here, force-probe molecular dynamics simulations were used to obtain insight into the tension-induced activation mechanism. We find evidence for a sequential mechanically induced opening of the catalytic site without complete domain unfolding. Our results suggest the rupture of two terminal beta-sheets as the primary unfolding steps. The low force resistance of the C-terminal relative to the N-terminal beta-sheet is attributed to their different geometry. A subsequent rearrangement of the autoinhibitory tail is seen to lead to the exposure of the active site, as is required for titin kinase activity. These results support the hypothesis of titin kinase as a force sensor.     

Mechanically induced sudden death in chest wall impact (commotio cordis)

Sudden death due to nonpenetrating chest wall impact in the absence of injury to the ribs, sternum and heart is known as commotio cordis. Although once thought rare, an increasing number of these events have been reported. Indeed, a significant percentage of deaths on the athletic field are due to chest wall impact. Commotio cordis is most frequently observed in young individuals (age 4–18 years), but may also occur in adults. Sudden death is instantaneous or preceded by several seconds of lightheadedness after the chest wall blow. Victims are most often found in ventricular fibrillation, and successful resuscitation is more difficult than expected given the young age, excellent health of the victims, and the absence of structural heart disease. Autopsy examination is notable for the lack of any significant cardiac or thoracic abnormalities.

In an experimental model of commotio cordis utilizing anesthetized juvenile swine, ventricular fibrillation can be produced by a 30 mph baseball strike if the strike occurred during the vulnerable period of repolarization, on the upslope of the T-wave. Energy of the impact object was also found to be a critical variable with 40 mph baseballs more likely to cause ventricular fibrillation than velocities less or greater than 40 mph. In addition, more rigid impact objects and blows directly over the center of the chest were more likely to cause ventricular fibrillation. Peak left ventricular pressure generated by the chest wall blow correlated with the risk of ventricular fibrillation. Activation of the K⁺_ATP channel is a likely cause of the ventricular fibrillation produced by chest wall blows. Successful resuscitation is attainable with early defibrillation.     

Mechanically induced electrical responses in murine mammary epithelial cells in primary culture

In mouse mammary epithelial cells in primary culture, mechanical stimulation of a cell induced in other cells within the same colony a short depolarization of less than 15 mV with a duration of 1-8 s and a subsequent, prominent hyperpolarization of 6 mV lasting 10-40 s. Epidermal growth factor induces a spontaneous hyperpolarizing response in cultured mammary cells, and in cells treated with EGF mechanical stimulation produced a greater hyperpolarization, while the amplitude of the depolarizing response was not affected. The amplitude of the mechanically induced hyperpolarization was markedly reduced by quinine and tetraethylammonium, blockers of the Ca2+ -dependent K+ channel. The results suggest that the Ca2+ -dependent K+ channel was involved in the hyperpolarization.     

MreB actin-mediated segregation of a specific region of a bacterial chromosome

Faithful chromosome segregation is an essential component of cell division in all organisms. The eukaryotic mitotic machinery uses the cytoskeleton to move specific chromosomal regions. To investigate the potential role of the actin-like MreB protein in bacterial chromosome segregation, we first demonstrate that MreB is the direct target of the small molecule A22. We then demonstrate that A22 completely blocks the movement of newly replicated loci near the origin of replication but has no qualitative or quantitative effect on the segregation of other loci if added after origin segregation. MreB selectively interacts, directly or indirectly, with origin-proximal regions of the chromosome, arguing that the origin-proximal region segregates via an MreB-dependent mechanism not used by the rest of the chromosome.     

MLN64 is involved in actin-mediated dynamics of late endocytic organelles

MLN64 is a late endosomal cholesterol-binding membrane protein of an unknown function. Here, we show that MLN64 depletion results in the dispersion of late endocytic organelles to the cell periphery similarly as upon pharmacological actin disruption. The dispersed organelles in MLN64 knockdown cells exhibited decreased association with actin and the Arp2/3 complex subunit p34-Arc. MLN64 depletion was accompanied by impaired fusion of late endocytic organelles and delayed cargo degradation. MLN64 overexpression increased the number of actin and p34-Arc-positive patches on late endosomes, enhanced the fusion of late endocytic organelles in an actin-dependent manner, and stimulated the deposition of sterol in late endosomes harboring the protein. Overexpression of wild-type MLN64 was capable of rescuing the endosome dispersion in MLN64-depleted cells, whereas mutants of MLN64 defective in cholesterol binding were not, suggesting a functional connection between MLN64-mediated sterol transfer and actin-dependent late endosome dynamics. We propose that local sterol enrichment by MLN64 in the late endosomal membranes facilitates their association with actin, thereby governing actin-dependent fusion and degradative activity of late endocytic organelles.     

Extracellular fusion of macrophage phagosomes with lysosomes

Following ingestion of micro-organisms by phagocytes, lysosomes fuse with the resulting phagocytic vacuoles. This process is presumed to assist in the destruction of the intracellular organisms. We have previously studied morphological and biochemical correlates of this subcellular fusion in macrophages and now report the observation of a similar fusion between cell-free organelles. Evidence so far obtained indicates that this fusion closely resembles its intracellular counterpart.     

A modular design for the clathrin- and actin-mediated endocytosis machinery

Endocytosis depends on an extensive network of interacting proteins that execute a series of distinct subprocesses. Previously, we used live-cell imaging of six budding-yeast proteins to define a pathway for association of receptors, adaptors, and actin during endocytic internalization. Here, we analyzed the effects of 61 deletion mutants on the dynamics of this pathway, revealing functions for 15 proteins, and we analyzed the dynamics of 8 of these proteins. Our studies provide evidence for four protein modules that cooperate to drive coat formation, membrane invagination, actin-meshwork assembly, and vesicle scission during clathrin/actin-mediated endocytosis. We found that clathrin facilitates the initiation of endocytic-site assembly but is not needed for membrane invagination or vesicle formation. Finally, we present evidence that the actin-meshwork assembly that drives membrane invagination is nucleated proximally to the plasma membrane, opposite to the orientation observed for previously studied actin-assembly-driven motility processes.     

Biogenesis of Afipia-containing phagosomes in non-professional phagocytes

Afipia felis is a Gram-negative alpha-proteobacterium, a rare cause of human cat scratch disease (CSD), and likely a pathogen of amoeba. Here, we show that various members of the genus Afipia attach to and are taken up by various non-professional phagocytic mammalian cells (epithelial CHO, endothelial EA.hy926, epithelial HeLa, epithelial INT407 cells, endothelial HMEC-1, endothelial HUVEC, and fibroblast L929 cells). However, only A. felis was able to do this efficiently. Invasion depended on a functional actin cytoskeleton and much less on microtubule dynamics. Bacteria were slowly taken up into HMEC-1 (and HUVEC) via pocket-like structures and they resided within membrane-surrounded phagosomes. While A. felis was found in a non-canonical endocytic compartment in macrophage cells, Afipia-containing phagosomes in HMEC-1 were transiently positive for early endosomal EEA1 and then became and remained positive for lysosome-associated membrane protein-1 (LAMP1) and the proton-pumping ATPase, suggesting undisturbed, albeit slowed, phagosome biogenesis in these cells. Similarly, at 24h of infection, most phagosomes in HeLa, INT407, HUVEC and in EA.hy926 cells were positive for LAMP1. In summary, A. felis enters various non-professional phagocytes and its compartmentation differs between macrophages and non-professional phagocytes.     

Gamma-secretase is a functional component of phagosomes

Gamma-secretase is a high molecular mass protein complex that catalyzes the intramembrane cleavage of its protein substrates. Two proteins involved in phagocytosis, CD44 and the low density lipoprotein receptor-related protein, are gamma-secretase substrates, suggesting that this complex might regulate some aspects of phagocytosis. Our results indicate that the four components of gamma-secretase, viz. presenilin, nicastrin, APH-1, and PEN-2, are present and enriched on phagosome membranes from both murine macrophages and Drosophila S2 phagocytes. The gamma-secretase components form high molecular mass complexes in lipid microdomains of the phagosome membrane with the topology expected for the functional enzyme. In contrast to the majority of the phagosome proteins studied so far, which appear to associate transiently with this organelle, gamma-secretase resides on newly formed phagosomes and remains associated throughout their maturation into phagolysosomes. Finally, our results indicate that interferon-gamma stimulates gamma-secretase-dependent cleavages on phagosomes and that gamma-secretase activity may be involved in the phagocytic response of macrophages to inflammatory cytokines.