bluuebunny/crossref_metadata_2025_split huggingface dataset:
Distribution of myosin and relationship to actin organization in cortical and subcortical areas of antibody‐labelled, quick‐frozen, deep‐etched fibroblast cytoskeletons
Authors: Lawson Durward 
Published: February 2005

AbstractIn this study I describe the ultrastructural distribution of myosin in cortical and subcortical areas of antibody‐labelled, quick‐frozen fibroblasts. In many cells myosin was present in small variably spaced and sized (0.23–0.39 μm long), nonaligned patches, while in other cells much larger periodically spaced patches of more uniform length (0.27 μm) were found. In all regions of the cytoskeleton myosin was found, primarily on linear bundles of actin filaments running parallel to the cell's long axis.Myosin was absent from single actin filaments, actin Filaments perpendicular to actin bundles aligned with the cell's long axis, and actin filaments, such as geodome vertices and parts of the cortex, which had a complex interwoven appearance. These data indicate that in motile non‐muscle cells myosin exerts force only in a unidirectional manner. Recognisable myosin filaments were never observed even in cells incubated either in N‐ethylmaleimide or sodium azide. The presence of myosin in, and almost to the very edge of, the cortex suggests that the cellular control of actomyosin based movement is direct and over short‐range distances. Large numbers of small cross‐linking filaments were found in association with cortical and subcortical actin. Their relationship to myosin and overall actin geometry is discussed.
Subcellular compartmentalization of phosphorylated neurofilament polypeptides in neurons
Authors: Hart Clyde E. , Nuckolls Glen H. , Wood John G. 
Published: February 2005

AbstractImmunocytochemistry and polyacrylamide gel electrophoresis have been used to study the distribution of phosphorylated forms of neurofilament antigens in rat brain. Immunostaining of tissue with an antisera produced against phosphatasesensitive domains of the 200‐kilodalton (kd) neurofilament polypeptide showed that phosphorylated forms of this polypeptide were present in virtually all axons and certain somata and dendrites of neurons in different brain regions. Immunoblots of whole brain homogenate or a neurofilament preparation from rat revealed that the affinity‐purified anti‐200‐kd sera used to immunostain tissue labeled the neurofilament‐associated 200‐kd band in a phosphatase‐sensitive manner. Fine structural analysis of this immunoreactivity in tissue showed that whenever the labeled organelle could be identified, it was a microtubule. In contrast, immunoblot analysis of twice‐cycled microtubules from porcine brain revealed that microtubules in vitro did not possess the 200‐kd antigen that was observed in situ. The results suggest that our antibody recognizes a phosphorylated domain on the neurofilament involved in cross‐linking neurofilaments and microtubules, and that in vivo, phosphorylated epitopes of the 200‐kd neurofilament polypeptide are capable of associating with microtubules.
Intracellular acidification, guanine‐nucleotide binding proteins, and cytoskeletal actin
Authors: Molski T. F. P. , Sha'afi R. I. 
Published: February 2005

AbstractThe addition of propionic acid to rabbit neutrophils causes cell acidification and increases the amount of actin associated with the cytoskeleton. Both responses are rapid, and while the cell acidification is somewhat long‐lasting, the increase in cytoskeletal actin is transient. It reaches a maximum value within 15 seconds and then return to the basal level. Unlike fMet‐Leu‐Phe, however, propionic acid does not cause a rise in the intracellular concentration of free calcium. Pretreatment of the cells with pertusis toxin inhibits the propionic acid‐produced increase in cytoskeletal actin but not the decrease in intracellular pH. However, the rate of return to the base line of the cell acidification produced by propionic acid is diminished in cells pretreated with pertussis toxin. On the other hand, both the decrease in intracellular pH and the increase in cytoskeletal actin produced by fMet‐Leu‐Phe are inhibited by pertussis toxin treatment. The results presented here suggest two important points. First, while cell acidification may trigger directly or indirectly the association of actin with the cytoskeleton, it is certainly not sufficient. Second, a functional guanine‐nucleotide regulatory protein is required for stimulated cytoskeletal actin. One or more components of the G‐protein and/or their effects on phosphoinositide hydrolysis may increase the number of actin monomers and the availability of preexisting actin filaments to these monomers.
Dynamics of behaviour during neuronal morphogenesis in culture
Authors: Jacobs J. Roger , Stevens John K. 
Published: February 2005

AbstractWe report a developmental sequence in the type and frequency of behaviours of neurons differentiating in vitro. We characterised these changes with extensive analysis of time‐lapse sequence from both the continuing cell line phenochromocytoma PC12 and primary mixed cell culture of cat and mouse central nervous system. PC12 cells activated by nerve growth factor (NGF) differentiate in a uniform and synchronous manner. This allowed the first quantification of changes in different neuron behaviours during morphogenesis.Shortly after NGF activation, PC12 cells are highly labile in morphology and exhibit a large variety of morphological behaviours. During the first week of differentiation, the frequency of these behaviours declines, and gross morphology becomes more stable. The frequency of neurite initiation after 1 week in NGF is one‐seventh what it was after 2 days in NGF. Over the same period, neurite retraction declines to one‐third, and somal migration ceases altogether. Growthcone activity does not decline during development. These behaviour changes correlate with published data on the differentiation of the neurite cytoskeleton.A qualitatively similar ontogeny was noted in the differentiation of CNS neurons in mixed cell culture. Major differnces occur in the relative timing of changes in behaviours. Mature, stable morphology is not detected in these cultures until 7 weeks in vitro.
Microinjected carboxylated beads move predominantly poleward in sea urchin eggs
Authors: Wadsworth Patricia 
Published: February 2005

AbstractObservations on living mitoic cells have suggested that material in the spindle moves poleward during mitosis. In order to investigate this movement, sea urchin eggs have been microinjected with 0.25‐μm diameter carboxylated fluorescent beads. When fluorescent beads were injected into unfertilized Lytechinus variegatus eggs, no motility was detected. When injected into mitotic cells, beads moved to the spindle poles. Individual beads moved rapidly, in a saltory fashion, and followed generally linear paths. Beads appeared to move along astral fibers, were generally excluded from thespindle proper, and accumulated at the spindle poles. Some dispersion of the beads away from the pole was observed as cells completed mitosis, but the majority of beads retained a polar location. After depolymerization of spindle microtubules with nocodazole, some dispersion of beads into the cytoplasm was also observed. Beads moved along taxol‐induced astral microtubules and accumulated at astral centers. These observations reveal that negatively chargedbeads accumulate rapidly at mitotic centers, moving toward the minus end of the microtubules. Neither the bidirectional motility of similar beads in interphase cells nor the plus‐end‐directed bead motility seen in axons was observed in these mitotic cells.
Fluorescence microscopy study of polymorphonuclear leukocyte substrate attached materials
Authors: Francis Joseph W. , Fabi Alain Y. , Petty Howard R. 
Published: February 2005

AbstractWe describe a technique to visualize substrate‐attached materials (SAM) of polymorphonuclear leukocytes (PMN) using the fluorescent lipid analog 1, 1′‐dioctadecyl‐3,3,3′,3′,‐tetramethylindocarbocyanine‐perchlorate (DiC18Icc). DiC18Icc was incorporated into the membranes of living cells or SAMs. Since cell preparation does not require fixation, SAMs can be rapidly visualized by fluorescence microscopy. SAMs are generated by subjecting attached cells to a shearing force by rinsing with phosphate‐buffered saline (PBS). The SAM‐labeling protocol identified a membrane compartment as shown by detergent extraction. The SAMs of PMN leukocytes observed with this technique display complex patterns of interconnecting filaments, foci with radiating filaments, and smooth membranous areas with interconnecting filaments. The sensitivity and nondestructive nature of the DiC18Icc‐labeling procedure have allowed us to observe filopodia of motile cells. The results are consistent with the hypothesis that locomotion involves a series of attachment and detachment steps. After 60 minutes of locomotion, these trailing filopodia have been measured at lengths up to 100 μm. The amount of membrane associated with these filopodia accounts for roughly 10% of the total membrane are of resting cells. These data set limits for models of membrane flow during chemotaxis.
Adaptation in the motility response to camp in dictyostelium discoideum
Authors: Varnum‐Finney Barbara , Schroeder Noel A. , Soll David R. 
Published: February 2005

AbstractWhen developing amebae of Dictyostelium discoideum are treated with constant concentrations of cAMP above 10−8 M, the average rate of motility is depressed, with maximum inhibition at roughly 10−6 M. It is demonstrated that shifting the concentration of cAMP from 0 M to concentrations ranging from 10−8 to 10−6 M in a perfusion chamber results in the immediate inhibition of motility. After shifting from 0 M to 10−8 or 10−7 M, the rate of cell motility remains low, then rebounds to a higher level, exhibiting a standard adaptation response. No adaptation is exhibited after a shift from 0 M to 10−6 M, a concentration resulting in maximum inhibition. It is demonstrated that the level of inhibition and the extent of the adaptation period are dependent upon the concentration of cAMP after the shift, and that submaximal inhibition is additive. The characteristics of adaptation in this motility response are very similar to the characteristics of adaptation for the relay system and phosphorylation of the putative cAMP receptor.
Actin filament patterns in mouse lens epithelium: A study of the effects of aging, injury, and genetics
Authors: Liou Willisa , Rafferty Nancy S. 
Published: February 2005

AbstractUsing mainly fluorescence microscopy after rhodamine‐phalloidin staining, the F‐actin distribution in the mouse lens epithelium was studied with regard to the effects of age, genetic strain, and mechanical injury.These studies have revealed that aside from its association with the plasma membrane the structural organization of F‐actin in the mouse lens epithelium in situ is characterized by two major configurations: (1) a filamentous arrangement in such patterns as stress fibers, polygonal arrays (PAs), and meshworks, and (2) a highly concentrated structure called a sequestered actin bundle (SAB).The aging study indicated that the SAB is a consistent character in C57BL/6 mice from the age of 5 wk on, but not in CF1 mice. The size and shape of the SAB change gradually with age as inferred from two‐dimensional measurements. The genetic study on the SAB character using hybrids and congenic strains showed that it is inherited as a Mendelian dominant, probably multigenic mode. Finally, the injury study revealed a structural modification in cells around the wound, including flattening of cells at the edge and extension of processes into the wound space. In the rest of the epithelium, injury amplified membrane infolding and fluorescence of polygonal arrays but diminished the size and fluorescence intensity of SABs. These changes are thought to be correlated with wound repair involving cell division and migration.These studies illustrate the variability in F‐actin expression in situ in lens epithelial cells that can be induced by intrinsic and extrinsic factors.
Axonal shortening and the mechanisms of axonal motility
Authors: George E. B. , Schneider B. F. , Lasek R. J. , Katz M. J. 
Published: February 2005

AbstractAxons in tissue culture retract and shorten if their tips are detached from the substrate. The shortening reaction of the axon involves contractile forces that also arise during normal axonal motility, elongation, and retraction. We studied shortening in axonal segments isolated from their parent axons by transecting the axon between the growth cone and the most distal point of adhesion to the substrate. Within 15–20 minutes after transection, an isolated axonal segment shortened and pulled its tail end toward the growth cone. During the shortening process, long sinusoidal bends arose along the axon. The identical shortening reaction occurs without transection, when the axon tip is detached from the substrate. Pharmacological studies with inhibitors of glycolysis indicate that the shortening mechanisms utilize metabolic energy, presumably ATP. The rate of sinusoidal shortening is similar to both the rate of polymer translocation in the axon by slow axonal transport and the rate of normal axonal elongation. Taxol inhibits the shortening reaction with a similar dose dependence to its inhibition of axonal growth. Together, all these observations suggest that the same basic intracellular motility mechanisms are involved in normal axonal growth, in slow axonal transport, and in the shortening reaction: the intracellular dynamic system that utilizes ATP to generate longitudinal movements of polymers within the axon may be the same mechanism underlying both the retraction and the elongation of the axon.
Video motion analysis of mitotic events in living cells of the fungus fusarium solani
Authors: Aist James R. , Bayles Carol J. 
Published: February 2005

AbstractAn earlier, laser microbeam study produced evidence that, in Fusarium solani, extranuclear polar forces function at anaphase B of mitosis to pull apart the incipient daughter nuclei, whereas the central spindle functions primarily to limit the rate at which they separate. To elucidate further the various dynamics of mitotic anaphase, 8–14 mitoses in hyphae of F. solani were analyzed at 0.5–2.0‐sec intervals using high‐resolution, digitally processed, videotaped sequences. The spindle growth rate, although fluctuating frequently, averaged 0.6 μm/min during metaphase, increased to 3.6 μm/min during anaphase A and was maximal at 6.1 μm/min during anaphase B. Commonly, chromosomes migrated poleward during anaphase A at fluctuating rates, the average rate being an unprecedented 7.5 μm/min. During anaphase the mitotic apparatus migrated to and fro in the hyphae at rates of 3–15 μm/min, an apparent effect of opposing, fluctuating and typically unequal cytoplasmic forces applied to the two spindle poles. Thus, the molecular mechanisms underlying the various anaphase movements in F. solani do not operate entirely smoothly and uniformly. Accelerated growth of the central spindle is temporally associated with anaphase A and the development of asters. Thus, chromosome disjunction may allow the polar forces to increase the rate of spindle elongation. Microtubule dynamics and motor molecules appear to be adequate to account for the observed rates of mitotic movements.
Progress in video microscopy
Authors: Inoué Shinya 
Published: February 2005

AbstractThe progress in video microscopy is reviewed from its early inception, especially with respect to improvements of the microscope image quality. Very recent advances that provide serial optical sections and depth of field as thin as 0.1 μm and that make possible the recording of birefringent images of individual microtubules (25 nm in diameter) directly in live, dividing cells are also documented.
“DMS,” a computer‐assisted system for quantitating motility, the dynamics of cytoplasmic flow, and pseudopod formation: Its application to Dictyostelium chemotaxis
Authors: Soll David R. 
Published: February 2005

AbstractA computer‐assisted Dynamic Morphology System (DMS) is described that allows the rapid quantitation of more than 30 parameters of motility and dynamic morphology for up to 40 amebae in parallel. This system also generates “difference pictures” for characterizing the dynamics of pseudopod formation. A 3‐D DMS is described, and application of DMS to problems of motility and chemotaxis in normal and mutant cells of Dictyostelium discoideum is reviewed.
The organization and regulation of the macrophage actin skeleton
Authors: Hartwig John H. , Yin Helen L. 
Published: February 2005

AbstractTo move, leukocytes extend portions of their cortical cytoplasm as pseudopods. These pseudopods are filled with a three‐dimensional actin filament skeleton, the reversible assembly of which in response to receptor stimulation is thought to play a major role in providing the mechanical force for these protrusive movements. The organization of this actin skeleton occurs at different levels within the cell, and a number of macrophage proteins have been isolated and shown to affect the architecture, assembly, stability, and length of actin filaments in vitro. The architecture of cytoplasmic actin is regulated by proteins that cross‐link filaments in higher‐order structures. Actin‐binding protein plays a major role in defining network structure by cross‐linking actin filaments into orthogonal networks. Gelsolin may have a central role in regulating network structure. It binds to the sides of actin filaments and severs them, and binds the “barbed” filament end, thereby blocking monomer addition at this end. Gelsolin is activated to bind actin filaments by μM calcium. Dissociation of gelsolin bound on filament ends occurs in the presence of the polyphosphoinositides, PIP and PIP2. Calcium and PIP2 have been shown to be intracellular messengers of cell stimulation.
Direct observation of mitotic spindle elongation in vitro
Authors: Baskin Tobias I. , Cande W. Zacheus 
Published: February 2005

AbstractSuccessful reactivation in vitro of anaphase B has recently been achieved with mitotic spindles isolated from the diatom Stephanopyxis turris. When a population of isolated spindles was studied indirectly by using immunofluorescence, nearly all of them were found to have elongated; however, when studied directly by using video microscopy, only a small proportion of spindles elongated. We report here conditions that allow nearly all of the spindles to elongate when observed directly with video microscopy. These direct observations validate previous ones made using indirect immunofluorescence. In addition, we find that the isolated spindles elongate with a linear rate, that the elongation is unchanged after the chromatin surrounding the spindles is digested with DNase I, and that during elongation a phase‐dense matrix may accumulate in the spindle midzone.
High hydrostatic pressure effects in vivo: Changes in cell morphology, microtubule assembly, and actin organization
Authors: Bourns Brenda , Franklin Samuel , Cassimeris Lynne , Salmon E. D. 
Published: February 2005

AbstractWe present the first study of the changes in the assembly and organization of actin filaments and microtubules that occur in epithelial cells subjected to the hydrostatic pressures of the deep sea. Interphase BSC‐1 epithelial cells were pressurized at physiological temperature and fixed while under pressure. Changes in cell morphology and cytoskeletal organization were followed over a range of pressures from 1 to 610 atm. At atmospheric pressure, cells were flat and well attached. Exposure of cells to pressures of 290 atm or greater caused cell rounding and retraction from the substrate. This response became more pronounced with increased pressure, but the degree of response varied within the cell population in the pressure range of 290–400 atm, Microtubule assembly was not noticeably affected by pressures up to 290 atm, but by 320 atm, few microtubules remained. Most actin stress fibers completely disappeared by 290 atm. High pressure did not simply induce the overall depolymerization of actin filaments for, concurrent with cell rounding, the number of visible microvilli present on the cell surface increased dramatically. These effects of high pressure were reversible. Cells re‐established their typical morphology, microtubule arrays appeared normal, and stress fibers reformed after approximately 1 hour at atmospheric pressure. High pressure may disrupt the normal assembly of microtubules and actin filaments by affecting the cellular regulatory mechanisms that control cytological changes during the transition from interphase into mitosis.
Long‐term analysis of organelle translocation in isolated axoplasm of Myxicola infundibulum
Authors: Breuer Anthony C. , Eagles Peter A. M. , Lynn Marc P. , Atkinson Mark B. , Gilbert Susan P. , Weber Leigh , Leatherman Judy , Hopkins John M. 
Published: February 2005

AbstractMoving intra‐axonal organelles demonstrate frequent variations in speed when viewed over several seconds. To evaluate these and other motion variations, a long‐term analysis of organelle motion in isolated axoplasm of Myxicola infundibulum was carried out using differential interference contrast optics and analog and digital image enhancement techniques. Motion characteristics of individual organelles were analyzed for periods of up to 58 minutes. Three principle observations on organelle motion were made: (1) Classes of organelles of the same size demonstrated a 5‐ to 25‐fold variation of speed, with the slowest speeds occurring most frequently; (2) organelle speeds over individual translocations (motion without stopping) are inversely proportional to their size, but the speeds calculated for the long‐term analysis of organelle motion (total distance travelled/total observation time, including pauses) did not reflect this observation; and (3) organelles displayed variable trip lengths, durations, mean speeds, and pause durations, and the relationships between these variations showed no repetitive patterns. In contrast to reported observations of uniform velocities of organelles moving on isolated microtubule preparations, these observations suggest that a variety of factors must play a role in organelle translocation in Myxicola axoplasm.
Tubulin tyrosination in Crithidia: Modifying enzymes and modification states of tubulin
Authors: Chang Sulie , Flavin Martin 
Published: February 2005

AbstractAn enzyme that adds C‐terminal tyrosine to tubulin has been identified in Crithidia fasciculata. It tyrosinates Crithidia, but not brain, tubulin and is specific for the α chain. Crithidia cells could not be shown to fix tyrosine in the absence of protein synthesis, which is consistent with the pattern of distribution of C‐terminal tyrosine in tubulin from different subcellular compartments of this protozoan. Terminal tyrosine was present in about 5% of flagellar α chain from cells in stationary phase and 20% from cells from midlog phase; none was detected in tubulin from cytosol or the subpellicular corset. In contrast to mammalian cells, in which a higher state of tyrosinolation characterizes recently assembled or unstable microtubules, terminal tyrosine was present only in the most stable polymer, the flagellar doublet microtubules.