Loss Of Actin Filaments Research Articles

Improved longevity of actomyosin in vitro motility assays for sustainable lab-on-a-chip applications

AbstractIn the in vitro motility assay (IVMA), actin filaments are observed while propelled by surface-adsorbed myosin motor fragments such as heavy meromyosin (HMM). In addition to fundamental studies, the IVMA is the basis for a range of lab-on-a-chip applications, e.g. transport of cargoes in nanofabricated channels in nanoseparation/biosensing or the solution of combinatorial mathematical problems in network-based biocomputation. In these applications, prolonged myosin function is critical as is the potential to repeatedly exchange experimental solutions without functional deterioration. We here elucidate key factors of importance in these regards. Our findings support a hypothesis that early deterioration in the IVMA is primarily due to oxygen entrance into in vitro motility assay flow cells. In the presence of a typically used oxygen scavenger mixture (glucose oxidase, glucose, and catalase), this leads to pH reduction by a glucose oxidase-catalyzed reaction between glucose and oxygen but also contributes to functional deterioration by other mechanisms. Our studies further demonstrate challenges associated with evaporation and loss of actin filaments with time. However, over 8 h at 21–26 °C, there is no significant surface desorption or denaturation of HMM if solutions are exchanged manually every 30 min. We arrive at an optimized protocol with repeated exchange of carefully degassed assay solution of 45 mM ionic strength, at 30 min intervals. This is sufficient to maintain the high-quality function in an IVMA over 8 h at 21–26 °C, provided that fresh actin filaments are re-supplied in connection with each assay solution exchange. Finally, we demonstrate adaptation to a microfluidic platform and identify challenges that remain to be solved for real lab-on-a-chip applications.

Nerve growth factor promotes reorganization of the axonal microtubule array at sites of axon collateral branching.

The localized debundling of the axonal microtubule array and the entry of microtubules into axonal filopodia are two defining features of collateral branching. We report that nerve growth factor (NGF), a branch-inducing signal, increases the frequency of microtubule debundling along the axon shaft of chicken embryonic sensory neurons. Sites of debundling correlate strongly with the localized targeting of microtubules into filopodia. Platinum replica electron microscopy suggests physical interactions between debundled microtubules and axonal actin filaments. However, as evidenced by depolymerization of actin filaments and inhibition of myosin II, actomyosin force generation does not promote debundling. In contrast, loss of actin filaments or inhibition of myosin II activity promotes debundling, indicating that axonal actomyosin forces suppress debundling. MAP1B is a microtubule associated protein that represses axon branching. Following treatment with NGF, microtubules penetrating filopodia during the early stages of branching exhibited lower levels of associated MAP1B. NGF increased and decreased the levels of MAP1B phosphorylated at a GSK-3β site (pMAP1B) along the axon shaft and within axonal filopodia, respectively. The levels of MAP1B and pMAP1B were not altered at sites of debundling, relative to the rest of the axon. Unlike the previously determined effects of NGF on the axonal actin cytoskeleton, the effects of NGF on microtubule debundling were not affected by inhibition of protein synthesis. Collectively, these data indicate that NGF promotes localized axonal microtubule debundling, that actomyosin forces antagonize microtubule debundling, and that NGF regulates pMAP1B in axonal filopodia during the early stages of collateral branch formation.

NOD2 promotes renal injury by exacerbating inflammation and podocyte insulin resistance in diabetic nephropathy

An increasing number of clinical and animal model studies indicate that activation of the innate immune system and inflammatory mechanisms are important in the pathogenesis of diabetic nephropathy. Nucleotide-binding oligomerization domain containing 2 (NOD2), a member of the NOD-like receptor family, plays an important role in innate immune response. Here we explore the contribution of NOD2 to the pathogenesis of diabetic nephropathy and found that it was upregulated in kidney biopsies from diabetic patients and high-fat diet/streptozotocin-induced diabetic mice. Further, NOD2 deficiency ameliorated renal injury in diabetic mice. In vitro, NOD2 induced proinflammatory response and impaired insulin signaling and insulin-induced glucose uptake in podocytes. Moreover, podocytes treated with high glucose, advanced glycation end-products, tumor necrosis factor-α, or transforming growth factor-β (common detrimental factors in diabetic nephropathy) significantly increased NOD2 expression. NOD2 knockout diabetic mice were protected from the hyperglycemia-induced reduction in nephrin expression. Further, knockdown of NOD2 expression attenuated high glucose-induced nephrin downregulation in vitro, supporting an essential role of NOD2 in mediating hyperglycemia-induced podocyte dysfunction. Thus, NOD2 is one of the critical components of a signal transduction pathway that links renal injury to inflammation and podocyte insulin resistance in diabetic nephropathy.

Subsynaptic AMPA Receptor Distribution Is Acutely Regulated by Actin-Driven Reorganization of the Postsynaptic Density

AMPA receptors (AMPARs) mediate synaptic transmission and plasticity during learning, development, and disease. Mechanisms determining subsynaptic receptor position are poorly understood but are key determinants of quantal size. We used a series of live-cell, high-resolution imaging approaches to measure protein organization within single postsynaptic densities in rat hippocampal neurons. By photobleaching receptors in synapse subdomains, we found that most AMPARs do not freely diffuse within the synapse, indicating they are embedded in a matrix that determines their subsynaptic position. However, time lapse analysis revealed that synaptic AMPARs are continuously repositioned in concert with plasticity of this scaffold matrix rather than simply by free diffusion. Using a fluorescence correlation analysis, we found that across the lateral extent of single PSDs, component proteins were differentially distributed, and this distribution was continually adjusted by actin treadmilling. The C-terminal PDZ ligand of GluA1 did not regulate its mobility or distribution in the synapse. However, glutamate receptor activation promoted subsynaptic mobility. Strikingly, subsynaptic immobility of both AMPARs and scaffold molecules remained essentially intact even after loss of actin filaments. We conclude that receptors are actively repositioned at the synapse by treadmilling of the actin cytoskeleton, an influence which is transmitted only indirectly to receptors via the pliable and surprisingly dynamic internal structure of the PSD.

Antibody to Hsp70 alters response of rabbit preimplantation embryos to hyperthermia in vitro

The aim of the study was to determine a role of Hsp70 in the response of rabbit preimplantation embryos to hyperthermia (HT) in vitro. The embryos were cultured at standard (ST, 37.5 °C, control) or elevated (HT, 41.5 °C) temperature for 6 h. In half of the embryos from both groups, Hsp70 was blocked by the addition of an antibody against Hsp70 into the medium at stages either prior to (≤8 cell) or after (morula) developmental block (DB). After 6 h, the HT embryos were post-cultured at 37.5 °C for 16–20 h and then evaluated for developmental stages, apoptosis (TUNEL, T), cell number and quality of actin cytoskeleton. The exposure of embryos to 41.5 °C either before or after DB did not affect blastocysts (Bl) rate, T-index and embryo cell number. Blockage of Hsp70 before DB resulted in no embryos at the Bl stage. From the Hsp70-blocked embryos after DB, only 27.6% (ST + anti-Hsp70) and 22.6% (HT + anti-Hsp70) of blastocysts were formed, versus 71.4% (ST) and 69.7% (HT) in the non-Hsp70-blocked group. T-index of Hsp70-blocked embryos prior to DB increased from 6.8% (ST) to 28.6% (ST + anti-Hsp70) and from 6.1% (HT) to 29.6% (HT + anti-Hsp70), whilst the cell number dropped from 111 ± 8.1 (ST) to 20.5 ± 2.42 (ST + anti-Hsp70) and from 106.8 ± 8.4 (HT) to 15.4 ± 1.34 (HT + anti-Hsp70). In contrast, in the Hsp70-blocked embryos after DB, neither T-index nor cell number was changed comparing to the non-blocked. Damages to the actin cytoskeleton, like intracytoplasmic clumps or loss of actin filaments, were only minimally caused by hyperthermia but mostly were a result of Hsp70 blockage. In conclusion, the embryos at stages before the genome activation (GA) appear to be more sensitive to HT. The resistance of pre-GA embryos to HT is related to Hsp70, whilst after GA, Hsp70 is probably not the only factor of thermotolerance and the existence of an additional protective mechanism(s) is hypothesized.

Comparative study of cyanotoxins affecting cytoskeletal and chromatin structures in CHO-K1 cells

In this study we compared the effects of the two frequently occuring and most dangerous cyanobacterial toxins on the cellular organization of microfilaments, microtubules and on the chromatin structure in Chinese hamster ovary (CHO-K1) cells. These compounds are the widely known microcystin-LR (MC-LR) and cylindrospermopsin (CYN) classified as the highest-priority cyanotoxin. Toxic effects were tested in a concentration and time dependent manner. The hepatotoxic MC-LR did not cause significant cytotoxicity on CHO-K1 cells under 20 μM, but caused apoptotic changes at higher concentrations. Apoptotic shrinkage was associated with the shortening and loss of actin filaments and with a concentration dependent depolymerization of microtubules. No necrosis was observed over the concentration range (1–50 μM MC-LR) tested. Cylindrospermopsin did cause apoptosis at low concentrations (1–2 μM) and over short exposure periods (12 h). Necrosis was observed at higher concentrations (5–10 μM) and following longer exposure periods (24 or 48 h). Cyanotoxins also affected the chromatin structure. The condensation process was inhibited by MC-LR at a later stage and manifested as broken elongated prechromosomes. CYN inhibited chromatin condensation at the early fibrillary stage leading to blurred fluorescent images of apoptotic bodies and preventing the formation of metaphase chromosomes. Cylindrospermopsin exhibited a more pronounced toxic effect causing cytoskeletal and nuclear changes as well as apoptotic and necrotic alterations.

Protein Synthesis in Distal Axons Is Not Required for Growth Cone Responses to Guidance Cues

Recent evidence suggests that growth cone responses to guidance cues require local protein synthesis. Using chick neurons, we investigated whether protein synthesis is required for growth cones of several types to respond to guidance cues. First, we found that global inhibition of protein synthesis stops axonal elongation after 2 h. When protein synthesis inhibitors were added 15 min before adding guidance cues, we found no changes in the typical responses of retinal, sensory, and sympathetic growth cones. In the presence of cycloheximide or anisomycin, ephrin-A2, slit-3, and semaphorin3A still induced growth cone collapse and loss of actin filaments, nerve growth factor (NGF) and neurotrophin-3 still induced growth cone protrusion and increased filamentous actin, and sensory growth cones turned toward an NGF source. In compartmented chambers that separated perikarya from axons, axons grew for 24-48 h in the presence of cycloheximide and responded to negative and positive cues. Our results indicate that protein synthesis is not strictly required in the mechanisms for growth cone responses to many guidance cues. Differences between our results and other studies may exist because of different cellular metabolic levels in in vitro conditions and a difference in when axonal functions become dependent on local protein synthesis.

IQGAP2 is required for the cadherin-mediated cell-to-cell adhesion in Xenopus laevis embryos

We have previously identified two Xenopus homologues of mammalian IQGAP, XIQGAP1 and XIQGAP2, which show high homology with human IQGAP1 and IQGAP2, respectively. In order to clarify function of the IQGAPs during development, we performed knock-down experiments on the XIQGAPs in Xenopus laevis embryos by microinjecting morpholino antisense oligonucleotides into blastomeres at the two-cell stage. Suppression of XIQGAP2 expression caused ectodermal lesions in the neurula stage embryos. While suppression of XIQGAP1 expression alone did not show any obvious defect in subsequent developmental processes, simultaneous knock-down of both XIQGAPs caused the ectodermal lesions during the gastrula stage. Histological studies suggested that a loss of cell adhesion in the ectodermal and mesodermal layers of the embryos caused the defect. The suppression of XIQGAP2 expression resulted in loss of actin filaments, β-catenin, and XIQGAP1 from cell borders in the ectoderm, although it did not affect the expression levels of these proteins. Furthermore, it inhibited Ca 2+-induced reaggregation of embryonic cells which had been dissociated in a Ca 2+/Mg 2+-free medium. These results strongly suggest that XIQGAP2 is crucial for cell adhesion during early development in Xenopus.

Green Tea Extract Modulates Actin Remodeling via Rho Activity in an In vitro Multistep Carcinogenic Model

Alteration of actin polymerization and loss of actin filaments is a marker of cellular dedifferentiation and early malignant transformation. To study this phenomenon, an in vitro human urothelial model consisting of two cell lines, HUC-PC and MC-T11, were incorporated into the study design. These two cell lines have different malignant transformation potential. The effect of green tea extract (GTE), a potential anticancer agent, on actin remodeling was investigated. Upon exposure to the carcinogen 4-aminobiphenyl (4-ABP), the untransformed HUC-PC undergoes malignant transformation whereas the transformed MC-T11 progresses from noninvasive to invasive tumor. GTE induces actin polymerization in MC-T11 cells in a dose-responsive manner, but this effect is less obvious in the untransformed, more differentiated HUC-PC cells, which natively have higher actin polymerization status. In contrast, GTE antagonizes carcinogen 4-ABP induced actin depolymerization and stress fiber disruption in HUC-PC cells. In MC-T11 cells, GTE inhibits 4-ABP induced motility by increasing cell adhesion and focal adhesion complex formation. The effect of GTE on actin remodeling seems to be mediated by the stimulation of small GTP-binding protein Rho activity, because C3 exoenzyme, a specific inhibitor for Rho, blocks GTE-mediated Rho activation and stress fiber formation in MC-T11 cells. This study shows that GTE exerts an effect on cytoskeletal actin remodeling and provides further support for the use of GTE as a chemopreventive agent.

Mechanical Properties of Membrane Surface of Cultured Astrocyte Revealed by Atomic Force Microscopy

In order to examine the mechanical properties of the membrane surface of astrocytes, we observed living astrocytes by atomic force microscopy (AFM) both in contact mode and force-mapping mode. Ridge-like structures reflecting actin filaments were observed in the topographic images in contact mode, but not in force-mapping mode, using a zero-loading force. When we measured the elasticity of astrocytes, we observed that the cell membrane above the nucleus was soft and the cell membrane above the cytosol was stiff. In particular, the parts reflecting actin filaments were very stiff. This effect of actin filaments on the elasticity of astrocytes was confirmed by the loss of actin filaments after application of actin-polymerization inhibitor.

Tyrphostin A9 and wortmannin perturb the Golgi complex and block proliferation of vascular smooth muscle cells

To proliferate, vascular smooth muscle cells first convert from a contractile to a synthetic phenotype. Earlier studies indicate that this process is supported by fibronectin and accelerated by platelet-derived growth factor (PDGF). Here, the mechanisms in this transition were further explored. Isolated rat aortic smooth muscle cells were treated with tyrphostin A9, a PDGF receptor tyrosine kinase inhibitor, and wortmannin, a phosphoinositide 3-kinase inhibitor. Electron microscopy did not show any effect on the reorganization of the cells during the first days in culture, i.e. the loss of actin filaments and the formation of a large secretory apparatus. Conversely, both drugs caused hypertrophy of the Golgi complex, with large and partly vacuolized cisternal stacks. Nevertheless, a juxtanuclear staining pattern for the Golgi enzyme mannosidase H, the coat protein α-COP, and the PDGF β-receptor was retained. Moreover, the serum-induced proliferation of the cells was blocked. These findings suggest that signaling via PDGF receptor tyrosine kinases and phosphoinositide 3-kinases is not necessary for the shift of the smooth muscle cells from a contractile to a synthetic phenotype On the other hand, these enzymes apparently carry out important functions in the control of intracellular membrane traffic and cell division.

Phenotypic modulation of smooth muscle cells after arterial injury is associated with changes in the distribution of laminin and fibronectin.

Earlier in vitro studies suggest opposing roles of laminin and fibronectin in regulation of differentiated properties of vascular smooth muscle cells. To find out if this may also be the case in vivo, we used immunoelectron microscopy to study the distribution of these proteins during formation of intimal thickening after arterial injury. In parallel, cell structure and content of smooth muscle alpha-actin was analyzed. The results indicate that the cells in the normal media are in a contractile phenotype with abundant alpha-actin filaments and an incomplete basement membrane. Within 1 week after endothelial denudation, most cells in the innermost layer of the media convert into a synthetic phenotype, as judged by loss of actin filaments, construction of a large secretory apparatus, and destruction of the basement membrane. Some of these cells migrate through fenestrae in the internal elastic lamina and invade a fibronectin-rich network deposited on its luminal surface. Within another few weeks a thick neointima forms, newly produced matrix components replace the stands of fibronectin, and a basement membrane reappears. Simultaneously, the cells resume a contractile phenotype, recognized by disappearance of secretory organelles and restoration of alpha-actin filaments. These findings support the notion that laminin and other basement membrane components promote the expression of a differentiated smooth muscle phenotype, whereas fibronectin stimulates the cells to adopt a proliferative and secretory phenotype.

Effect of tumour necrosis factor, heat, and radiation on the viability and microfilament organization in cultured endothelial cells.

Normal blood vessels are leakage proof, non-adherent to blood cell elements, and participate actively in directional blood flow. These properties rely on the shape of endothelial cells and the integrity of the endothelial cell monolayer. The often observed effects of tumour necrosis factor-alpha (TNF) and hyperthermia on tumour tissue are the disruption of blood flow and an increase of vascular permeability. These agents are also known to affect the cytoskeletal organization and the cytoskeleton-dependent cellular functions. We observed that TNF (100 U/ml for 60 min) or heat (43 degrees C for 60 min) treatment causes the collapse of actin filaments in human umbilical vein endothelial cells (HUVEC). The combined treatment of TNF and hyperthermia intensifies the change of shape and loss of actin filaments. However, these changes are reversible within 24 h. These transient changes may contribute to the dysfunction and increased leakage of the microvasculature in tumours during and after these treatments despite the fact that the viability determined by MTT assay did not show a significant interaction between TNF and hyperthermia. Radiation (5 Gy) and TNF interact to a lesser extent compared with heat and TNF on cell shape and actin filament organization in HUVEC. Heat or radiation treatment enhances the expression of ELAM-1 mRNA in HUVEC while TNF produces the strongest effect on ELAM-1 mRNA expression. Our study suggests that radiation and heat affect endothelial cells and their subsequent functions differently. Result of an interaction between heat and TNF on endothelial cells supports the common notion that the anti-tumour effect by heat plus TNF treatments may benefit due to the increased disruption of vasculature function in the tumour.