Modification of the bi-directional sliding movement of actin filaments along native thick filaments isolated from a clam

A reliable viability assay for Giardia is required for the development of disinfection process design criteria and pathogen monitoring by water treatment utilities. Surveys of single-staining nucleic acid dyes (stain dead parasites only), and double-staining vital dye kits from Molecular Probes (stain live and dead parasites) were conducted to assess the viability of untreated, heat-killed, and chemically inactivated Giardia muris cysts. Nucleic acid staining results were compared to those of in vitro excystation and animal infectivity. Nucleic acid stain, designated as SYTO-9, was considered the best among the single-staining dyes for its ability to stain dead cysts brightly and its relatively slow decay rate of visible light emission following DNA binding. SYTO-9 staining was correlated to animal infectivity. A Live/Dead BacLight was found to be the better of 2 double-staining viability kits tested. Logarithmic survival ratios based on SYTO-9 and Live/Dead BacLight were compared to excystation and infectivity results for G. muris cysts exposed to ozone or free chlorine. The results indicate that SYTO-9 and Live/Dead BacLight staining is stable following treatment of cysts with chemical disinfectants.     

Mutation of cis-proline 207 in mitochondrial creatine kinase to alanine leads to increased acid stability

SETTING: Hamad General Hospital, the tertiary health centre for Doha, Qatar. OBJECTIVE: The purpose of the study was to define and correlate the role of radiology with clinical and pathological findings in abdominal tuberculosis. MATERIAL AND METHODS: A total of 59 patients (47 males and 12 females) diagnosed bacteriologically and/ or histologically for abdominal tuberculosis were radiologically assessed. Evaluation was based on the analysis of plain abdominal radiographs, gastro-intestinal contrast studies (barium meal follow through and barium enema), ultrasonography and computed tomography. RESULTS: Plain abdominal radiographs performed in 38 patients were positive in 19 cases (50%). Gastrointestinal contrast examinations were positive in 27 out of 34 cases (80%). Ultrasound examinations were abnormal in 25 out of 31 cases (81%), while computed tomography, performed in 24 patients, revealed abnormal findings in 19 cases (80%). Combined radiographic and imaging procedures revealed peritoneal involvement (ascites) in 16 patients (27%), bowel involvement in 36 (61%), mass lesion in 11 (19%), lymphadenopathy in 13 (22%) and organ involvement in 13 (22%). CONCLUSION: There was no single radiological method that provided all necessary information suggestive of abdominal tuberculosis. Although unequivocal diagnosis of abdominal tuberculosis can only be made by culture and histological findings, combined computed tomography and ultrasound findings were the most important imaging tools in the diagnostic process for abdominal tuberculosis, while contrast studies helped to assess the extent of bowel disease, hence influencing decisions concerning surgery.     

Both microtubules and actin filaments are required for efficient postendocytotic traffic of the polymeric immunoglobulin receptor in polarized Madin-Darby canine kidney cells

It has been postulated that membrane traffic in polarized epithelial cells requires both actin filaments and microtubules. We have tested this hypothesis by analyzing the effect of cytochalasin D (cytoD; an actin-disrupting agent), by itself or in combination with nocodazole (a microtubule depolymerizing agent), on postendocytic traffic in Madin-Darby canine kidney cells. CytoD treatment inhibited basolateral to apical transcytosis of IgA in polymeric immunoglobulin receptor-expressing cells by approximately 45%, but had little effect on basolateral recycling of transferrin. Apical recycling of IgA was also inhibited by approximately 20%. Like nocodazole, cytoD acted at an early step in transcytosis, and inhibited translocation of IgA between the basolateral early endosomes and the apical recycling endosome. There was little inhibition of the subsequent release of IgA from the apical recycling endosome of cytoD- or nocodazole-treated cells. Order-of-addition experiments suggest that the cytoD-sensitive step preceded the nocodazole-sensitive step. Treatment with both cytoD and nocodazole inhibited transcytosis 95%. These results suggest that in addition to microtubules, efficient postendocytic traffic in polarized epithelial cells also requires actin filaments.     

How to analyze electron micrographs of rafts of actin filaments crosslinked by actin-binding proteins

Actin bundles are common cytoskeletal structures but ones which are usually polymorphic, varying from bundle to bundle. Two-dimensional arrays of actin filaments crosslinked by actin-bundling proteins are more tractable structures to analyze than are the three-dimensional bundles found in cells. The first step in analyzing these two-dimensional "rafts" is to determine the spatial relationships between neighboring filaments. It is difficult to discern such relationships by inspection of the electron micrographs of rafts, but easy by examination of the Fourier transforms. We provide theory and examples of the analysis of transforms of rafts, and show that different bundling proteins give rise to different kinds of rafts.     

Organization of actin and microtubules during process formation in tau-expressing Sf9 cells

Insect Sf9 cells usually elaborate a highly characteristic single process when infected with a baculovirus encoding recombinant human tau. The processes are unbranched, of uniform caliber, and contain bundles of microtubules. Because taxol treatment alone does not induce process outgrowth in these cells, it is believed that tau confers properties on microtubules that permits the conversion of microtubule assembly into the formation of processes. Here we have analyzed the reorganization of both actin filaments and microtubules during process initiation. A zone of organelle exclusion representing the focal reorganization of actin at one pole of the cell anticipated process emergence. A relationship between actin organization and process emergence was also suggested by a shift from single to multiple process formation after treatment with cytochalasin D. The rate of process elongation doubled after cytochalasin treatment of tau-expressing cells. The increase in rate was due to the inhibition of the growth arrest phases which occur in the absence of cytochalasin. In contrast, Sf9 cells treated with cytochalasin after more than 20 h of tau expression were relatively resistant to the drug's effects. We conclude that actin and microtubules are specifically reorganized during tau-induced process outgrowth and that a dynamic relationship between actin filaments and microtubules effects process formation.     

Model for the alignment of actin filaments in endothelial cells subjected to fluid shear stress

Cultured vascular endothelial cells undergo significant morphological changes when subjected to sustained fluid shear stress. The cells elongate and align in the direction of applied flow. Accompanying this shape change is a reorganization at the intracellular level. The cytoskeletal actin filaments reorient in the direction of the cells' long axis. How this external stimulus is transmitted to the endothelial cytoskeleton still remains unclear. In this article, we present a theoretical model accounting for the cytoskeletal reorganization under the influence of fluid shear stress. We develop a system of integro-partial-differential equations describing the dynamics of actin filaments, the actin-binding proteins, and the drift of transmembrane proteins due to the fluid shear forces applied on the plasma membrane. Numerical simulations of the equations show that under certain conditions, initially randomly oriented cytoskeletal actin filaments reorient in structures parallel to the externally applied fluid shear forces. Thus, the model suggests a mechanism by which shear forces acting on the cell membrane can be transmitted to the entire cytoskeleton via molecular interactions alone.     

Coronin promotes the rapid assembly and cross-linking of actin filaments and may link the actin and microtubule cytoskeletons in yeast

Coronin is a highly conserved actin-associated protein that until now has had unknown biochemical activities. Using microtubule affinity chromatography, we coisolated actin and a homologue of coronin, Crn1p, from Saccharomyces cerevisiae cell extracts. Crn1p is an abundant component of the cortical actin cytoskeleton and binds to F-actin with high affinity (Kd 6 x 10(-9) M). Crn1p promotes the rapid barbed-end assembly of actin filaments and cross-links filaments into bundles and more complex networks, but does not stabilize them. Genetic analyses with a crn1Delta deletion mutation also are consistent with Crn1p regulating filament assembly rather than stability. Filament cross-linking depends on the coiled coil domain of Crn1p, suggesting a requirement for Crn1p dimerization. Assembly-promoting activity is independent of cross-linking and could be due to nucleation and/or accelerated polymerization. Crn1p also binds to microtubules in vitro, and microtubule binding is enhanced by the presence of actin filaments. Microtubule binding is mediated by a region of Crn1p that contains sequences (not found in other coronins) homologous to the microtubule binding region of MAP1B. These activities, considered with microtubule defects observed in crn1Delta cells and in cells overexpressing Crn1p, suggest that Crn1p may provide a functional link between the actin and microtubule cytoskeletons in yeast.     

Apolar growth of Neurospora crassa leads to increased secretion of extracellular proteins

Protein secretion in filamentous fungi has been shown to be restricted to actively growing hyphal tips. To determine whether an increase in the amount f growing surface area of a fungus can lead to an increase in the amount of protein secretion, we examined secretion in a temperature-sensitive Neurospora crassa mcb mutant that shows a loss of growth polarity when incubated at restrictive temperature. Incubation of the mcb mutant at restrictive temperature results in a three- to fivefold increase in the level of extracellular protein and a 20-fold increase in carboxymethyl cellulase activity relative to a wild-type strain. A mutation in the cr-1 gene has been shown previously to suppress the apolar growth phenotype of the mcb mutant, and we find that the level of extracellular protein produced by a mcb; cr-1 double mutant was reduced to that of the wild-type control. Immunolocalization of a secreted endoglucanase revealed that proteins are secreted mainly at hyphal tips in hyphae exhibiting polar growth and over the entire surface area of bulbous regions of hyphae that are produced after a shift of the mcb mutant to restrictive temperature. These results support the hypothesis that secretion of extracellular protein by a filamentous fungus can be significantly increased by mutations that alter growth polarity.     

Relationship of actin, microtubules, and crosswall synthesis during septation in Aspergillus nidulans

A significant limitation of neural networks is that the representations they learn are usually incomprehensible to humans. We have developed an algorithm, called TREPAN, for extracting comprehensible, symbolic representations from trained neural networks. Given a trained network, TREPAN produces a decision tree that approximates the concept represented by the network. In this article, we discuss the application of TREPAN to a neural network trained on a noisy time series task: predicting the Dollar-Mark exchange rate. We present experiments that show that TREPAN is able to extract a decision tree from this network that equals the network in terms of predictive accuracy, yet provides a comprehensible concept representation. Moreover, our experiments indicate that decision trees induced directly from the training data using conventional algorithms do not match the accuracy nor the comprehensibility of the tree extracted by TREPAN.     

Models for the length distributions of actin filaments: I. Simple polymerization and fragmentation

We studied mathematical models for the length distributions of actin filaments under the effects of polymerization/depolymerization, and fragmentation. In this paper, we emphasize the effects of these two processes acting alone. In this case, simple discrete and continuous models can be derived and solved explicitly (in several special cases), making the problem interesting from a modeling and pedagogical point of view. In a companion paper (Ermentrout and Edelstein-Keshet, 1998, Bull. Math. Biol. 60, 477-503) we investigate what happens when the processes act together, with particular attention to fragmentation by gelsolin, and with a greater level of biological detail.     

Role of the ninaC proteins in photoreceptor cell structure: ultrastructure of ninaC deletion mutants and binding to actin filaments

The ninaC proteins are found in Drosophila photoreceptor cells. Their primary sequences suggest they are kinase/myosin chimeras, but their myosin head-like domain is the most divergent amongst all the myosin-like proteins described to date. To investigate possible roles of the ninaC proteins in cell structure, we examined the ultrastructure of the photoreceptor cells in various ninaC mutants, and tested the ability of the proteins to interact with actin filaments in a myosin-like manner. In flies lacking the larger ninaC protein, p174, an ultrastructural phenotype was evident before eclosion. The axial actin cytoskeleton of the rhabdomeral microvilli appeared either fragmented or as an isolated structure, without linkage to the microvillar membrane. Deletion of the myosin head-like domain or the calmodulin-binding domain of p174 resulted in a similar abnormal cytoskeleton. Breakdown of the rhabdomeres followed, although at different rates depending on the deletion. Lack of the smaller protein, p132, per se did not result in photoreceptor degeneration, but in older flies there was an abnormal accumulation of multivesicular bodies. Moreover, the presence of p132 retarded the degeneration that occurs in the absence of p174, even though the p132 remained outside the rhabdomere. Biochemical studies showed that both ninaC proteins bind actin filaments and cosediment with actin filaments in an ATP-sensitive manner. These results outline structural roles for the ninaC proteins, and are consistent with the notion, suggested by their amino acid sequences, that the proteins are actin-based mechanoenzymes.     

Desmin and actin filaments in membrane-cytoskeletal preparations of the electric tissue of Electrophorus electricus, L

OBJECTIVES: To assess the effect of undertaking custodial care of a grandchild on grandparents' depression levels and to determine what characteristics are associated with higher depression levels among caregiving grandparents. DESIGN: A longitudinal national probability panel study: the National Survey of Families and Households. The first wave of data (n= 13 008) was collected in 1987 and 1988, and the second wave of data (n=10008) was collected from 1992 through 1994. SETTING: The survey was conducted in respondents' households in the coterminous United States. PARTICIPANTS: The subsample for this study was composed of 3111 respondents who reported being grandparents during the 1992-1994 interviews and for whom complete depression information was available. Of these grandparents, 158 were the primary caregivers for their grandchildren in the 1990s. MAIN OUTCOME MEASURES: Depression was measured using a modified version of the Center for Epidemiological Studies Depression Scale. RESULTS: Those who provide primary care for a grandchild are almost twice as likely to have levels of depressive symptoms above the traditional Center for Epidemiological Studies Depression Scale cut point of 16 (25.1% vs 14.5%). Even when controlling for baseline depression and demographic variables known to affect depressive symptoms, undertaking the care of a grandchild was associated significantly with higher depression levels in a multivariate prospective analysis (P<.01). Among caregiving grandparents, those who recently assumed caregiving responsibilities (P<.05) and women (P<.10) were more depressed and older respondents (P<.10) and those in good health (P<.001) were less depressed. CONCLUSIONS: Undertaking the primary care of a grandchild is associated with an increase in levels of depression. Particularly in light of the recent dramatic increase in the prevalence of grandparent caregiving in the United States, physicians need to explore familial role changes with midlife and older patients who have symptoms of depression. Special attention should be paid to the most at-risk subsets of grandparent caregivers: those who are new caregivers, those in poor health, those who are younger, and women.     

The organization and animal-vegetal asymmetry of cytokeratin filaments in stage VI Xenopus oocytes is dependent upon F-actin and microtubules

Confocal immunofluorescence microscopy with anti-cytokeratin antibodies revealed a continuous and polarized network of cytokeratin (CK) filaments in the cortex of stage VI Xenopus oocytes. In the animal cortex, CK filaments formed a dense meshwork that both was thicker and exhibited a finer mesh than the network of CK filaments previously observed in the vegetal cortex (Klymkowsky et al., 1987). CK filaments first appeared in association with germinal vesicle (GV) and mitochondrial mass (MM) of oocytes in early mid stage I, indicating that CK filaments are the last of the three cytoskeletal networks to be assembled. By late stage I, CK filaments formed complex networks surrounding the GV, surrounding and penetrating the MM, and linking these networks to a meshwork of CK filaments in the oocyte cortex. During stage III-early IV, CK filaments formed a highly interconnected, apparently unpolarized, radial array linking the perinuclear and cortical CK filament networks. Polarization of the CK filament network was observed during mid stage IV-stage V, as first the animal, then the vegetal CK filament networks adopted the organization characteristic of stage VI oocytes. Treatment of stage VI oocytes with cytochalasin B disrupted the organization of both cortical and cytoplasmic CK filaments, releasing CK filaments from the oocyte cortex and inducing formation of numerous cytoplasmic CK filament aggregates. CB also disrupted the organization of cytoplasmic microtubules (MTs) in stage VI oocytes. Disassembly of oocyte MTs with nocodazole resulted in loss of the characteristic A-V polarity of the cortical CK filament network. In contrast, disruption of cytoplasmic CK filaments by microinjection of anti-CK antibodies had no apparent effect on cytoplasmic or MT organization. We propose a model in which the organization and polarization of the cortical network of CK filaments in stage VI Xenopus oocytes are dependent upon a hierarchy of interactions with actin filaments and microtubules.     

Tropomyosin and troponin regulation of wild type and E93K mutant actin filaments from Drosophila flight muscle. Charge reversal on actin changes actin-tropomyosin from on to off state

In the Drosophila flight muscle actin mutant E93K there is a charge reversal on the surface of actin close to the proposed position of tropomyosin when it is in the off state. Using a quantitative in vitro motility assay we have found that the wild type Drosophila ACT88F actin behaved like rabbit skeletal muscle actin when tropomyosin and troponin were added at pCa5 and pCa9. In contrast the effect of tropomyosin upon the E93K mutant actin filament movement was completely different from wild type and resembled the response of wild type with tropomyosin+troponin at pCa9 (i.e. the filaments were switched off). Velocity of E93K actin did not increase, and the fraction of filaments motile was reduced to less than 15% by adding up to 30 nM tropomyosin. When myosin subfragment-1 modified by N-ethylmaleimide was mixed with mutant E93K actin-tropomyosin filaments we observed that it restored motility of the filaments to the level observed with E93K actin alone. We conclude that electrostatic charge on the surface of domain 2 of actin plays a critical role in determining the state of actin-tropomyosin that is a central feature of the steric blocking mechanism of actin filament regulation.     

Actin filaments and microtubules are involved in different membrane traffic pathways that transport sphingolipids to the apical surface of polarized HepG2 cells

In polarized HepG2 hepatoma cells, sphingolipids are transported to the apical, bile canalicular membrane by two different transport routes, as revealed with fluorescently tagged sphingolipid analogs. One route involves direct, transcytosis-independent transport of Golgi-derived glucosylceramide and sphingomyelin, whereas the other involves basolateral to apical transcytosis of both sphingolipids. We show that these distinct routes display a different sensitivity toward nocodazole and cytochalasin D, implying a specific transport dependence on either microtubules or actin filaments, respectively. Thus, nocodazole strongly inhibited the direct route, whereas sphingolipid transport by transcytosis was hardly affected. Moreover, nocodazole blocked "hyperpolarization," i.e., the enlargement of the apical membrane surface, which is induced by treating cells with dibutyryl-cAMP. By contrast, the transcytotic route but not the direct route was inhibited by cytochalasin D. The actin-dependent step during transcytotic lipid transport probably occurs at an early endocytic event at the basolateral plasma membrane, because total lipid uptake and fluid phase endocytosis of horseradish peroxidase from this membrane were inhibited by cytochalasin D as well. In summary, the results show that the two sphingolipid transport pathways to the apical membrane must have a different requirement for cytoskeletal elements.     

Changing patterns of localization of the tobacco mosaic virus movement protein and replicase to the endoplasmic reticulum and microtubules during infection

Tobacco mosaic virus (TMV) derivatives that encode movement protein (MP) as a fusion to the green fluorescent protein (MP:GFP) were used in combination with antibody staining to identify host cell components to which MP and replicase accumulate in cells of infected Nicotiana benthamiana leaves and in infected BY-2 protoplasts. MP:GFP and replicase colocalized to the endoplasmic reticulum (ER; especially the cortical ER) and were present in large, irregularly shaped, ER-derived structures that may represent "viral factories." The ER-derived structures required an intact cytoskeleton, and microtubules appeared to redistribute MP:GFP from these sites during late stages of infection. In leaves, MP:GFP accumulated in plasmodesmata, whereas in protoplasts, the MP:GFP was targeted to distinct, punctate sites near the plasma membrane. Treating protoplasts with cytochalasin D and brefeldin A at the time of inoculation prevented the accumulation of MP:GFP at these sites. It is proposed that the punctate sites anchor the cortical ER to plasma membrane and are related to sites at which plasmodesmata form in walled cells. Hairlike structures containing MP:GFP appeared on the surface of some of the infected protoplasts and are reminiscent of similar structures induced by other plant viruses. We present a model that postulates the role of the ER and cytoskeleton in targeting the MP and viral ribonucleoprotein from sites of virus synthesis to the plasmodesmata through which infection is spread.     

Increasing the dose and rate of Albunex infusion leads to superior left ventricular contrast effect

In routine clinical use, the efficacy of Albunex in producing clinically useful opacification may be lower than in initial clinical studies. We hypothesized that increasing either the rate of injection or amount of Albunex administered would increase left ventricular opacification. Fifty adult volunteers were each injected with Albunex in five volume/rate combinations. Blinded reviewers evaluated left ventricular opacification and endocardial border delineation compared with the baseline (noncontrast) echocardiogram. In addition, captured digitized images were analyzed with video-densitometric techniques. Injected at the highest volume/rate tested (20 ml at 3.0 ml/sec), Albunex provided the greatest improvement in left ventricular opacification, endocardial border delineation, and quality of the echocardiogram. The administration of Albunex caused no serious adverse events at any volume/rate regimen tested. Our data indicate that faster injection rates and larger dose volumes than those currently recommended by the package insert significantly improve Albunex ultrasound contrast without compromising safety.