bluuebunny/crossref_metadata_2025 huggingface dataset:
Slow pulsatile movements of Schwann cells in vitro: A time‐lapse cinemicrographic study
Authors: Forman David S. , Shain William G. , Fuchs David A.
Published: February 2005

Slow pulsatile movements of Schwann cells in vitro were studied quantitatively by using time‐lapse cinemicrography. Schwann cells from peripheral nerves of 3‐day‐old rats were cultured in serum‐free medium. Most Schwann cells showed intermittent episodes of pulsatile movement; each episode consisted of one or several contractile pulses. About half of the episodes consisted of a single pulse, and episodes with more than four pulses were rare. The average episode of activity lasted 2.6 min, while the average duration of a single pulse was 1.5 min. The mean quiescent interval between episodes of activity was 3.7 min. Some cells showed no pulsatile activity. Active cells averaged 6.6 episodes/h. The fraction of time which a Schwann cell spent in pulsatile activity varied widely, with an average of 28%. Behavior of Schwann cells in HEPES‐buffered Hanks saline was generally similar to that in the complete medium. Raising K+ to 40 mM or Ca++ to 10 mM did not markedly affect the time course of the pulsatile motility, although the contractions were more vigorous in the high Ca++. Pulsatile movement was reversibly inhibited by cytochalasin B and appeared to be potentiated by drugs that disrupt microtubules.
Theoretical analysis of radioactivity profiles during fast axonal transport: Effects of deposition and turnover
Authors: Reed M. C. , Blum J. J.
Published: February 2005

In a preceding study [Blum, J.J., and Reed, M.C. (1985): Cell Motil. 5:507–527], factors responsible for the shape and velocity of the leading edge of the radiolabeled organelle profile were analyzed, but processes that might influence the shape of the plateau‐like region behind the advancing wave were ignored. It is now shown that deposition of material from the fast transport system into membrane‐associated structures, degradation of such deposited material and its return to the soma by the retrograde transport system, or leakage of radiolabeled material from the axon can account for the shape of the plateau. Furthermore, these processes are compatible with the maintenance of such structural inhomogeneities as the nodes of Ranvier.
Substructure of sidearms on squid axoplasmic vesicles and microtubules visualized by negative contrast electron microscopy
Authors: Langford George M. , Allen Robert D. , Weiss Dieter G.
Published: February 2005

We present a high‐resolution electron microscopic study of the sidearms on microtubules and vesicles that are suggested to form the crossbridges which produce the microtubule‐based vesicle transport in squid axoplasm. The sidearms were found attached to the surfaces of the anterogradely transported vesicles in the presence of ATP. These sidearms were made of one to three filaments of uniform diameter. Each filament measured 5–6 nm in width and 30–35 nm in length. The filaments in some of the sidearms had splayed apart by pivoting at their base, thereby assuming a “V” shape. The spread configuration illustrated the independence of the individual filaments. The filaments in other sidearms were closely spaced and oriented parallel to each other, a pattern called the compact configuration. In axoplasmic buffer containing AMP‐PNP, structures indistinguishable from the filaments of the sidearms on the vesicles were observed attached to microtubules. Pairs of filaments, thought to represent the basic functional unit, were observed attached to adjacent protofilaments of the microtubules by their distal tips. These data support a model of vesicle movement in which a pair of filaments within a sidearm forms two crossbridges and moves a vesicle by “walking” along the protofilaments of the microtubule.
Polymorphic assembly of subtilisin‐cleaved tubulin
Authors: White Elizabeth A. , Burton Paul R. , Himes Richard H.
Published: February 2005

Limited proteolysis of tubulin with subtilisin results in cleavage of both the α and β subunits, releasing small peptides from the C‐terminal ends. At 37°C the digested tubulin assembles into polymorphic structures: microtubules with attached ribbons in the presence of GTP, rings in the presence of GDP, and protofilament spirals in the presence of vinblastine. Undigested tubulin does not assemble under these conditions. Rings and Vinca‐induced spiral structures are assembled from undigested tubulin only when microtubule‐associated proteins, high Mg2+ concentrations, or polycations are present. Thus, cleavage with subtilisin affects assembly in a manner similar to the addition of these agents. It appears that binding of positively charged substances may act by neutralizing the charge on the highly acidic C‐terminal regions of the α‐ and β‐subunits, while cleavage with subtilisin produces the same effect by removing these peptides. Undigested and subtilisin‐digested tubulin form sheets of protofilaments in the presence of Zn2+, which indicates that the binding sites for the 2–3 Zn2+ ions necessary to induce sheet formation do not reside in the C‐terminal regions of the monomers.
Contractility of the spasmoneme in glycerinated Vorticella stalk induced by various divalent metal and lanthanide ions
Authors: Yokoyama Yukiko , Asai Hiroshi
Published: February 2005

The glycerinated stalks of the peritrich ciliate, Vorticella, can contract helically and reversibly on the addition of not only Ca2+ but also other divalent or trivalent cations having ionic radii not far from 1 Å. In order to investigate the stalk contraction quantitatively in the absence of Ca2+‐chelators, we developed a method to eliminate contaminating Ca2+ and other metal ions in KC1 and pHbuffer solutions by using a Ca2+‐and heavy metal ion‐specific ion exchange resin (Eporas MX‐2) Thus, it was possible to measure the relationship between the fractional stalk length of Vorticella and the free concentration of alkaline earth metal, transition metal, and lanthanide ions in the 0.1 M KC1 and buffer (pH 6.8) solutions. Among these ions, Ca2+, Nd3+, and Eu3+ (having ionic radii of about 1 Å) had the highest affinity for the contractile element in the spasmoneme. As the concentration of lanthanide ions (except Nd3+ and Eu3+) is increased, the Vorticella stalk contracts abruptly at a threshold level; this means that the Hill's parameter is very high, probably more than 6. The results of these experiments and of the co‐mixtures of Ca2+ and Tb3+ suggest that a contractile element in the spasmoneme contains both contractile Ca2+‐binding and regulatory Ca2+‐binding sites.
Wave of cortical actin polymerization in the sea urchin egg
Authors: Yonemura Shigenobu , Mabuchi Issei
Published: February 2005

The distribution of actin filaments in the cortical layer of sea urchin eggs during fertilization has been investigated by light microscopy using fluorescently labeled phallotoxins. The cortical layer of both whole eggs and cortices isolated on a glass surface was examined. In cortices of unfertilized eggs, numerous fluorescent spots were seen, which may correspond to short actin filament cores in microvilli. After insemination, one of the sperm‐attaching points on the egg surface first became strongly fluorescent. This fluorescence grew around the point of sperm penetration with the growth of the fertilization cone. Then, the cortical layer of the egg around the fertilization cone became strongly fluorescent and the fluorescence propagated in a wavelike manner over the entire cortex. The mechanism of the propagation of actin polymerization is discussed.
Role of cortical tension in fibroblast shape and movement
Authors: Albrecht‐Buehler Guenter
Published: February 2005

In order to analyze the cellular mechanism of shape formation, the shape of individual 3T3 cells was perturbed by micromanipulation resulting in the detachment and relaxation of a cellular extension and the bending of the extension to form an “elbow” at a variable angle β. Finally, the tip of the extension was allowed to reattach to the substrate away from the cell.

The cells reacted by drawing the extension tight. If β < 90°, the elbow moved laterally for 8–15 min until the extension projected orthogonally at the cell surface. If β ≥90°, the extension remained stationary, Finally, in all cases webs formed between attachment points in the perturbed area. If the tip of the extension was allowed to touch its own cell body, thus forming a loop, the cells invariably closed the loop.

The paper interprets the cellular reaction as the result of cortical tension and suggests that it is a major factor in the formation of fibroblast shape and the expressions of fibroblast motility.
Reversible inhibition of microtubuies and cell growth by the bicyclic colchicine analogue MTC
Authors: Díez José C. , Avila Jesús , Nieto Juan M. , Andreu José M.
Published: February 2005

2‐methoxy‐5‐(2,3,4‐trimethoxyphenyl) 2,4,6‐cycloheptatrien‐1‐one (MTC) is a synthetic colchicine analogue, lacking the B ring of the alkaloid (Fitzgerald: Biochem. Pharmacol. 25:1381–1387, 1976). MTC has been shown to bind reversibly to the colchicine binding site of tubulin and to inhibit microtubule assembly in vitro (Andreu et al: Biochemistry 23:1742–1752, 1984; Bane et al: J. Biol. Chem. 259:7391–7398, 1984). Its action on different cultured cell lines (PtK2, Pk15, and SV‐3T3) has now been studied. 0.2 × 10−6 M MTC stopped Pkl5 and SV‐3T3 cell growth, inducing an accumulation of mitoses in a few hours. Removal of MTC from the culture medium rapidly restored normal mitotic index and growth rates. Partial depolymerization of the cytoplasmic microtubules of PtK2 cells was observed at concentrations ranging from 2 to 5 × 10−7 M. Maximal microtubule network depolymerization was obtained after 4 h of treatment with 2 to 5 × 10−6 M MTC or at a higher MTC concentration (2 × 10−5 M) for less than 2 h. Removal of 2 × 10−5 M MTC (the highest MTC concentration used) from the culture medium resulted in almost complete microtubule polymerization after 10 min of drug recovery and a normal microtubule network in 20–30 min.

MTC constitutes an antimitotic drug directed to the colchicine site. It is water‐soluble, shows a fast and reversible action, and may therefore be employed as a convenient tool to study cellular microtubule‐dependent functions.
Patterns of tubulin isotype synthesis and usage during mitotic spindle morphogenesis in Physarum
Authors: Paul Eileen C. A. , Roobol Anne , Foster Kay E. , Gull Keith
Published: February 2005

Tubulin synthesis in the naturally synchronous plasmodium of Physarum polycephalum is a markedly periodic event restricted to the late G2 period of the cell cycle. Mitosis in the plasmodium is intranuclear, and there are no cytoplasmic microtubules at any stage of the cell cycle. We have combined a biochemical investigation of the synthesis of the plasmodial tubulin isotypes and their participation in the mitotic spindle with a microscopic study (immunofluorescence) of the development of spindle microtubules throughout the cell cycle.

We have shown that all four tubulin isotypes identified in the plasmodium (α1, α2, β1 and β2) are present in the mitotic spindle. The stoichiometry of isotype usage in the mitotic spindle generally reflects the overall abundance of isotypes in the plasmodium as a whole: β2 > α1 > α2 > β1. We have also shown that tubulins synthesized in the G2 period of one cell cycle can be incorporated into the spindles of the immediately ensuing mitosis and have sufficient biological longevity to allow participation in the mitotic divisions of future cell cycles. Thus, the phenomenon of periodic tubulin synthesis does not reflect a restricted use of tubulin to the cell cycle in which it was synthesized. The major polymerization of tubulin in the nucleus occurred less than 30 min before metaphase. A novel tubulin‐containing structure was, however, present in the nucleus approximately 60 min before metaphase. Polymerized tubulin is rapidly removed from the nucleus following nucleokinesis.
Distribution of multiple centrospheres determines migration of BHK syncitia
Authors: Lewis Lindiann , Albrecht‐Buehler Guenter
Published: February 2005

After fusion of BHK cells with polyethylene glycol, the resulting syncitia contained in 77% of the cases multiple microtubule organizing centers (MTOCs), which were aggregated into a common centrosphere. Based on the observation of phagokinetic tracks, we found that the syncitia were able to locomote if (1) the MTOCs aggregated into a common centrosphere cluster, and (2) the clustered centrospheres were excluded from the cluster of nuclei of the syncitium. The results suggest that each individual pair of one nucleus and one centrosphere contributes, in a process of vectorial addition, its individual polarity to the polarity of the syneitium. Thus the widely accepted idea that the centrosphere is involved in the determinatinn of cell polarity can be generalized beyond the case of single cells.
Cordycepin disrupts the microtubule networks and arrests nil 8 hamster fibroblasts at the onset of mitosis
Authors: Zieve Gary W. , Feeney Robert J. , Roemer Elizabeth J.
Published: February 2005

The nucleoside analogue 3′‐deoxyadenosine (cordycepin) arrests dividing cells at the onset of mitosis in prometaphase. The microtubules in the arrested prometaphase cells depolymerize to two small asters. A minimum of 80 μg/ml cordycepin or 20 μg/ml cordycepin in combination with 2 μg/ml of the deaminase inhibitor erythro‐9‐(2‐hydroxy‐3‐nonyl) adenosine (EHNA) lo inhibit its degradation is required to see these effects. Analysis of cell extracts by high‐pressure liquid chromatography indicates that cordycepin enters the cells rapidly and is phosphorylated to 3′‐dATP. The intracellular concentration rises almost linearly from 0.7 mM after 15 min to 7 mM by 210 min. Concomitantly the ATP concentration shows a rapid drop from the 4 mM present in controls. However, the direct reduction of ATP levels does not mimic the same rapid effects of cordycepin on the microtubules. In addition, similar effects are not produced by a variety of other adenosine analogues with alterations in the 2′ and 3′ ribose positions. Although other pharmacological reagents arrest cells at the onset of mitosis, cordycepin is unusual because of the collapse of the microtubule networks to two small asters that radiate from the microtubule‐organizing center. 3′‐dATP can replace the requirement for ATP or GTP in the vitro polymerization of microtubules from microtubule protein: however, at limiting concentrations of nucleotide it requires approximately two times the concentration of 3′‐dATP as ATP to support an equivalent level of microtubule polymerization. This suggests that the effects of cordycepin in vivo may be the result of the depletion of cellular ATP pools and the altered ability of 3′dATP to substitute for ATP‐dependent reactions. Current experiments are testing this hypothesis.
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

In 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

Immunocytochemistry 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

The 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

We 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

Observations 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

We 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

When 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

Using 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

Axons 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

An 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

The 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

A 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

To 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

Successful 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

We 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

Moving 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

An 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.
EGTA induces prolonged summed depolarizations in Mytilus gill coupled ciliated epithelial cells: Implications for the control of ciliary motility
Authors: Stommel E. W. , Stephens R. E.
Published: February 2005

Abfrontal ciliated cells of Mytilus edulis gill beat when mechanically stimulated, a consequence of a Ca++‐based generator potential and regenerative response. In contrast, the lateral ciliated epithelial cells arrest when stimulated, a consequence of a Ca++‐based generator potential and a Na+/Ca++‐based regenerative response. Iontophoretic injection of EGTA in abfrontal cells, followed by mechanical stimulation, results in a large, prolonged depolarization that returns to the resting level stepwise. It has been hypothesized that this phenomenon is caused by successive Ca++‐dependent repolarizations in coupled cells, first in adjacent cells and then in the injected cell, in accord with relative EGTA loading. We have now demonstrated this same stepwise repolarization phenomenon in the Na+/Ca++‐dependent lateral ciliated cells. In this case, each repolarization step is often preceded by a small spike. With either cell type, using two‐electrode recording techniques, we can detect the stepwise repolarization in distant cells, proportionately decremented when the second (KCl) electrode is some distance from the injection (EGTA) electrode and stimulus. When force is applied between the electrodes and nearest the KCl electrode, a greater initial response is recorded from this electrode, presumably resulting from depolarization of its impaled cell, prolonged by EGTA diffusion through the intervening cell junctions. The subsequent repolarization steps are of approximately the same size, suggesting repolarization of cells between the two electrodes. These observations are consistent with the cell coupling/EGTA loading hypothesis and indicate that both cell types mediate repolarization through Ca++ and propagate ciliary beat or arrest through intracellular coupling.
Localization of mitotic‐apparatus‐associated 51‐kD protein in unfertilized and fertilized sea urchin eggs
Authors: Ohta Kunihiro , Toriyama Masaru , Endo Sachiko , Sakai Hikoichi
Published: February 2005

The 51‐kD protein, a protein component of the mitotic apparatus in sea urchin eggs, is involved in the aster‐forming activity previously shown in vitro [Toriyama et al., 1988]. Postembedding immunofluorescent labelings of eggs from fertilization through first cleavage showed that the 51‐kD protein is localized in sperm asters, centrosomal regions, spindles, basal regions of astral microtubules, and regions surrounding daughter nuclei at telophase in situ. Immunofluorescence and immunoblot analyses detected the 51‐kD protein uniformly in unfertilized eggs, but not in spermatozoa. When unfertilized eggs were treated with taxol, the 51‐kD protein was shown to be associated with taxol‐induced cytasters. Immunoblot analysis revealed that similar protein species are present in the mitotic apparatus of other species of sea urchin. It was suggested that the 51‐kD protein may be involved in microtubule nucleation and microtubule matrix in sea urchin eggs in vivo.
B‐band protein in sea urchin sperm flagella
Authors: Inaba Kazuo , Mohri Toshiko , Mohri Hideo
Published: February 2005

A high‐molecular‐weight polypeptide, named B‐band, was partially purified from sea urchin sperm flagella using selective extraction, hydroxylapatite chromatography, and sucrose density gradient centrifugation. The molecular weight of the B‐band was 440,000 by continuous system of sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE). Sedimentation coefficient of the B‐band protein was 10.5 S, and its Stokes radius was 10 nm. When examined by low‐angle rotary shadowing electron microscopy, this molecule appeared to be composed of four globular heads and two curved linkers (“double headphone shape”), which was quite different from the shape of 21 S dynein, the outer arm dynein. Flagellar axonemes were also subjected to several chemical dissections. The B‐band was not extracted with treatments that remove both arm structures but was solubilized with treatments that extract other components such as radial spokes and nexin links. The B‐band protein in the axoneme was also more susceptible to trypsin digestion than the arm structures. These results suggest that the B‐band protein is a “double headphone‐shaped” component of the axonemal structures and makes up the elastic structure that might regulate the active sliding between adjacent doublet microtubules.
Ionic control of locomotion and shape of epithelial cells: II. Role of monovalent cations
Authors: Bereiter‐Hahn Jürgen , Vöth Monika
Published: February 2005

The migration of keratocytes isolated from Xenopus tadpole epidermis has been investigated in vitro. In saline the cells move with a mean speed of 5–6 μm/min. Migration is slowed down in saline with diminished sodium content and ceases in media containing not more than 4 mM sodium. Inhibition of the Na+/K+‐2Cl− cotransporter by piretanide reduces the speed of migrating cells to about one‐third of the control level, the same accounts to inhibition of the Na+/H+ antiport with amiloride at pH 7.2. At pH 6.6, however, amiloride only slightly influences locomotion. Depolarization of the plasma membrane by increased extracellular K+ concentration or by inhibition of the Na+/K+ pump by ouabain is only of minor influence during more than 1 h. Hyperpolarization of the cells using the sodium ionophore monensin impedes locomotion; this inhibition depends on an active Na+/K+ pump. Ionophore‐mediated breakdown of the K+ gradient strictly inhibits locomotion. The experiments have shown that a continuous flux of sodium ions is indispensable for the maintenance of cell locomotion. These ions may exert their action primarily by affecting cytosolic free calcium concentration and pH.
Maturation of hagfish gland thread cells: Composition and characterization of intermediate filament polypeptides
Authors: Spitzer Robert H. , Koch Elizabeth A. , Downing Stephen W.
Published: February 2005

Previous studies with the hagfish, a primitive vertebrate, have shown that the gland thread cells (GTCs) each contain a single thread (∼60 cm long in average‐sized cells) in the form of a concisely coiled cytoskeletal entity destined for export by holocrine secretion. The thread in relatively immature GTCs consists almost entirely of intermediate filaments (IFs) bundled in parallel alignment with far fewer microtubules (MTs). The three thread polypeptides described earlier (α, basic; β acidic; γ, most acidic; each with a Mr of 63–64 kD) are now further evaluated with respect to in vitro assembly, cross‐reactivity with IF polypeptides from higher vertebrates, and peptide sequence homology with known IF polypeptides. The overall results mainly suggest that the hagfish polypeptides are keratinlike substances but lamins or a new type of IF is not ruled out. However, cross‐reactivity is weak with mammalian keratins; the 8–11‐nm filaments formed from mixtures of α and γ in vitro are generally linear rather than the curvilinear structures usually formed by keratin and nonkeratin IFs; and mixtures of α and β tend to yield 9–12‐nm granules or granular strings. Polypeptide analyses on GTCs segregated on the basis of maturational stage show a progressive increase in β/γ values which correlates with cell maturation, but the α/(β+γ) ratios remain near 1. Inasmuch as β and γ have many similar properties, the documented increase in the amount of the β component in aging GTCs might in part be the result of a failure in a posttranslational modification system and may contribute to the ultrastructural changes that accompany thread maturation in preparation for holocrine secretion and subsequent modulation of the viscoelastic properties of mucus.
Electron spectroscopic imaging of the intercellular bridge
Authors: Rattner J. B. , Bazett‐Jones D. P.
Published: February 2005

Phosphorylated proteins have been implicated in the control and regulation of many cellular events. One of the striking features of microtubule organizing centers is that they are rich in phosphoproteins [Vandre et al., 1984]. In this report we describe the application of electron spectroscopic imaging to the study of the spatial and temporal distribution of phosphorus and hence to the study of phosphorylated proteins associated with the formation and organization of one such organizing center, the intercellular bridge. Our study indicates that there are several discrete microtubule‐based domains that ultimately come to lie within the intercellular bridge and that there is a unique pattern of phosphorylation–dephosphorylation in some of these domains. These patterns correlate with the concurrent growth and sliding of antiparallel microtubules and the development of interactions among neighboring microtubules.
Functional diversity among spectrin isoforms
Authors: Coleman Thomas R. , Fishkind Douglas J. , Mooseker Mark S. , Morrow Jon S.
Published: February 2005

The purpose of this review on spectrin is to examine the functional properties of this ubiquitous family of membrane skeletal proteins. Major topics include spectrin‐membrane linkages, spectrin‐filament linkages, the subcellular localization of spectrins in various cell types and a discussion of major functional differences between erythroid and nonerythroid spectrins. This includes a summary of studies from our own laboratories on the functional and structural comparison of avian spectrin isoforms which are comprised of a common alpha subunit and a tissue‐specific beta subunit. Consequently, the observed differences among these spectrins can be assigned to differences in the properties of the beta subunits.