bluuebunny/crossref_metadata_2025 huggingface dataset:
A quantitative study of the role of F‐actin in producing neutrophil shape
Authors: Watts Raymond G. , Crispens Marta A. , Howard Thomas H.
Published: February 2005

Neutrophils change shape from round to polar and sequentially polymerize/depolymerize actin following chemotactic peptide activation in suspension. To study the relationship between changes in F‐actin content and shape we altered the kinetics/extent of actin polymerization and depolymerization with tBOC peptide, cytochalasin D (CD), and low‐dose FMLP, and determined the effect of these alterations on the temporal sequence of changes in neutrophil shape. F‐actin was measured by FACS analysis of NBDphallacidin‐stained cells and expressed as relative fluorescent intensity (RFI) compared to control (RFI = 1.00). Shape was determined by scanning electron microscopy. FMLP causes serial polymerization/depolymerization of actin (RFI = 1.00 ± 0.04, 1.60 ± 0.21, 1.10 ± 0.18, and 1.05 ± 0.14) associated with four distinct shapes (round‐smooth, round‐ruffled, blebbed, and polar) noted at 0, 30, 90, 300 sec respectively. Since blebbed and polar shapes appear concurrent with depolymerization and following polymerization, we determined whether depolymerization is required for polarization of cells. The kinetics of depolymerization were: (1) accelerated by tBOC addition at 45 sec, and (2) slowed by high concentrations of FMLP (>10‐7 M) (300 sec RFI = 1.46). Neither change altered the time course of shape change. To determine whether duration of actin polymerization defines shape, polymerization was halted by addition of tBOC at 5, 10, 20, 30 sec after FMLP to block actin polymerization and shape was monitored at 300 sec. TBOC added 5–20 sec after FMLP limited neutrophil shape change to the blebbed form, while tBOC addition 30 sec following FMLP resulted in a polar shape at 300 sec. To determine whether the extent of actin polymerization affects the shape change sequence, polymerization was limited by (1) inhibition of polymerization with CD, (2) exposure of cells to low concentrations of FMLP ( < 10‐9 M), and (3) interruption of polymerization with tBOC. Actin polymerization to RFI < 1.35‐fold basal results in blebbed shape; polymerization > 1.35‐fold basal yields polar shape. The data show: (1) the human neutrophil demonstrates intermediate shapes when activated by chemotactic peptide, (2) depolymerization of F‐actin does not determine shape, and (3) blebbed shape appears when actin polymerizes for >5 sec; polar shape with polymerization ≥30 sec to RFI > 1.35‐fold basal. The data suggest actin polymerization is required for, and extent of polymerization determines, the shape of human neutrophils.
Metabolic regulation in mammalian sperm: Mitochondrial volume determines sperm length and flagellar beat frequency
Authors: Cardullo Richard A. , Baltz Jay M.
Published: February 2005

In the absence of glycolytic support, mammalian sperm derive their energy for motility from a densely packed array of mitochondria at the base of the flagellum known as the midpiece. Using data on the morphometric dimensions of over 200 mammalian species, we found that an allometric relationship exists between midpiece length (Lm) and flagellum length (Lf). Specifically, the length of the midpiece varies approximately as the 3/2 power of the flagellar length although the proportionality constant is different for eutherian and marsupial sperm. In contrast, when we corrected for the fraction of the midpiece that was taken up by mitochondria, a single linear correlation between mitochondrial volume and flagellar length for all mammals was found. These allometric relationships were used along with basic flagellar hydrodynamic theories to establish a unifying equation that predicted flagellar frequencies for any mammalian sperm between 40 μm and 200 μm in length. These findings imply that, at least in mammals, the mechanisms for energy production and dissipation in sperm flagella are highly conserved.
Ultrastructure of the human erythrocyte cytoskeleton and its attachment to the membrane
Authors: Ursitti Jeanine A. , Pumplin David W. , Wade James B. , Bloch Robert J.
Published: February 2005

We attached paraformaldehyde‐fixed human erythrocyte ghosts to coated coverslips and sheared them to expose the cytoskeleton. Quick‐freeze, deep‐etch, rotary‐replication, or tannic acid/osmium fixation and plastic embedding revealed the cytoskeleton as a dense network of intersecting straight filaments. Previous negative stain studies on spread skeletons found 5–6 spectrin tetramers intersecting at each actin oligomer, with an estimated 250 such intersections/μm2 of membrane. In contrast, we found 3–4 filaments at each intersection and ∼400 intersections/μm2 of membrane. Immunogold labeling verified that the filaments were spectrin, but their lengths (29–37 nm) were approximately one‐third that of extended spectrin dimers. The length and diameter of the filaments were sufficient to accommodate spectrin dimers, but not spectrin tetraments. Our results suggest that, in situ, spectrin dimers may associations as hexamers and octamers, rather than tetramers. We present several explanations that can reconcile our observations on intact cytoskeletons with previous reports on spread material.

Extracting sheared ghosts with solutions of low ionic strength removed the cytoskeleton to reveal projections from the cytoplasmic surface of the membrane. These projections contained band 3, as shown by immunogold labeling, and they aggregated to a similar extent as intramembrane particles (IMP) when the cytoskeleton was removed, suggesting a direct relationship between these structures. Quantification indicated a stoichiometry of 2 IMP for each cytoplasmic projection. Cytoplasmic projections presumably contain other proteins besides band 3 since further treatment with high ionic strength solutions extracts peripheral proteins and reduces the diameter of projections by ∼3 nm.
F‐actin bundling protein from Physarum polycephalum: Purification and its capacity for co‐bundling of actin filaments and microtubules
Authors: Itano Naoki , Hatano Sadashi
Published: February 2005

An F‐actin bundling protein was isolated and purified from plasmodium of Physarum polycephalum. The F‐actin bundling protein in Physarum extract was passed through a DEAE‐cellulose column. After the protein in the fraction was treated with 6 M urea, it was purified by gel filtration on Sephacryl S‐300 HR followed by chromatography on CM‐Toyopearl (cation exchange) in the presence of 6 M urea. The purified protein gave a single band on SDS‐PAGE, and the molecular weight was estimated to be 52,000. This F‐actin bundling protein is referred to as the 52 kDa protein.

Interestingly, the 52 kDa protein also induced bundling of microtubules. The formation of F‐actin and microtubule bundles was Ca2+‐insensitive, but depended on the salt concentration. Each bundle formed at NaCl concentrations less than 0.1 M. The 52 kDa protein cross‐reacted with monoclonal antibody raised against a HeLa 55 kDa protein (an F‐actin bundling protein from HeLa cells) (Yamashiro‐Matsumura and Matsumura: J. Biol. Chem. 260:5087‐5097, 1985).

When the 52 kDa protein was added to a mixture of actin filaments and microtubules, co‐bundles composed of both filaments formed. This is the first reported example in which an F‐actin bundling protein induced co‐bundling of actin filaments and microtubules.
Substrate‐bound factors stimulate engorgement of growth cone lamellipodia during neurite elongation
Authors: Burmeister Donald W. , Rivas Rodolfo J. , Goldberg Daniel J.
Published: February 2005

The surfaces on which neurons grow greatly affect neurite elongation, but it is unclear how substrates influence the events within the growth cone that bring about elongation. Neurite elongation by Aplysia californica neurons in culture occurs through a series of transformations of the structures of the growth cone (Goldberg and Burmeister, J. Cell Biol., 103:1921‐1931, 1986). The growth cone produces actin‐rich protrusions, veils, and lamellipodia, which can then mature into the central body of the growth cone through the net advance of microtubules and membranous organelles from contiguous central regions, a process called “engorgement.” Aplysia neurons form growth cones on poly‐l‐lysinetreated substrates, but their rate of neurite elongation is greatly enhanced on substrates additionally exposed to Aplysia hemolymph. The acute application of hemolymph to slowly growing neurites brings about a rapid acceleration of neurite elongation and engorgement. The enhancement of engorgement was effected with material eluted from hemolymph‐treated substrates and was not seen when hemolymph was added to neurons cultured on hemolymph‐treated substrates inactivated by exposure to UV radiation. Thus, we conclude that the rapid acceleration of engorgement caused by hemolmph is, in large part, a substrate‐mediated effect. We propose that extracellular substrate molecules can modulate the rate of neurite growth through the regulation of the engorgement of lamellipodia.

The microtubule disrupters colcemid and nocadazole inhibit the advance of vesicular elements into the lamellipodia following hemolymph treatment, but taxol, which promotes the polymerization and stabilization of microtubules, does not itself enhance engorgement. The microfilament disrupter cytochalasin B, however, stimulates engorgement. Our results suggest that regulating the resistance of the peripheral actin meshwork to penetration by microtubules and vesicles may be a mechanism by which substrate‐attached molecules regulate neurite advance.
Microtubule sliding in flagellar axonemes of Chlamydomonas mutants missing inner‐ or outer‐arm dynein: Velocity measurements on new types of mutants by an improved method
Authors: Kurimoto Eiji , Kamiya Ritsu
Published: February 2005

To help understand the functional properties of inner and outer dynein arms in axonemal motility, sliding velocities of outer doublets were measured in disintegrating axonemes of Chlamydomonas mutants lacking either of the arms. Measurements under improved solution conditions yielded significantly higher sliding velocities than those observed in a previous study [Okagaki and Kamiya, 1986, J. Cell Biol. 103:1895‐1902]. As in the previous study, it was found that the velocities in axonemes of wild type (wt) and a mutant (oda1) missing the outer arm differ greatly: 18.5 ± 4.1 μm/sec for wt and 4.4 ± 2.3 μm/sec for oda1 at 0.5 mM Mg‐ATP. In contrast, axonemes of two types of mutants (ida2 and ida4) that lacked different sets of two inner‐arm heavy chains displayed velocities almost identical with the wild‐type velocity. Moreover, axonemes of a non‐motile double mutant ida2 × ida4 underwent sliding disintegration at a similar high velocity, although less frequently than in axonemes of single mutants. These observations support the hypothesis that the inner and outer dynein arms in disintegrating axonemes drive microtubules at different speeds and it is the faster outer arm that determines the overall speed when both arms are present. The inner arm may be important for the initiation of sliding. The axoneme thus appears to be equipped with two (or more) types of motors with different intrinsic speeds.
The dynamic instability of microtubules is not modulated by α‐tubulin tyrosinylation
Authors: Idriss Haitham , Stammers David K. , Ross Carl K. , Burns Roy G.
Published: February 2005

The tyrosinylation of chick brain α‐tubulin and the effects of the tyrosinylation status on the assembly and dynamic instability of chick brain MAP2:tubulin microtubule protein have been examined. Each of the eight major α‐isotypes can be tyrosinylated in vitro, irrespective of whether a C‐terminal tyrosine is genetically encoded. The extent of tyrosinylation is however limited to ≏ 0.3 mol.mol−1. The tyrosinylation status (0 vs. 0.3 mol.mol−1) has no effect on either the assembly kinetics of chick brain microtubule protein or on the rate of length redistribution following assembly and shearing. It is therefore unlikely that the tyrosinylation status directly affects the intrinsic stability of assembled microtu‐bules since the rate of length redistribution is both a sensitive assay and a function of the kinetic parameters governing dynamic instability.
Epitope mapping of monoclonal antibodies against caldesmon and their effects on the binding of caldesmon to Ca++/calmodulin and to actin or actin‐tropomyosin filaments
Authors: Lin Jim Jung‐Ching , Davis‐Nanthakumar Elizabeth J. , Jin Jian‐Ping , Lourim David , Novy Robert E. , Lin Jenny Li‐Chun
Published: February 2005

The effects of monoclonal anti‐caldesmon antibodies, C2, C9, C18, C21, and C23, on the binding of caldesmon to F‐actin/F‐actin‐tropomyosin filaments and to Ca++/calmodulin were examined in an in vitro reconstitution system. In addition, the antibody epitopes were mapped by Western blot analysis of NTCB (2‐nitro‐5‐thiocyanobenzoic acid) and CNBr (cyanogen bromide) fragments of caldesmon. Both C9 and C18 recognize an amino terminal fragment composed of amino acid residues 19 to 153. The C23 epitope lies within a fragment ranging from residues 230 to 386. Included in this region is a 13‐residue repeat sequence. Interestingly this repetitive sequence shares sequence similarity with a sequence found in nuclear lamin A, a protein which is also recognized by C23 antibody. Therefore, it is likely that the C23 epitope corresponds to this 13‐residue repeat sequence. A carboxyl‐terminal 10K fragment contains the epitopes for antibodies C2 and C21. Among these antibodies, only C21 drastically inhibits the binding of caldesmon to F‐actin/F‐actin‐tropomyosin filaments and tc Ca++/calmodulin. When the molar ratio of monoclonal antibody C21 to caldesmon reached 1.0, a maximal inhibition (90%) on the binding of caldesmon to F‐actin filaments was observed. However, it required double amounts of C21 antibody to exhibit a maximal inhibition of 70% on the binding of caldesmon to F‐actin‐tropomyosin filaments. These results suggest that the presence of tropomyosin in F‐actin enhances caldesmon's binding. Furthermore, C21 antibody also effectively inhibits the caldesmon binding to Ca++/calmodolin. The kinetics of C21 inhibition on caldesmon's binding to Ca++/calmodulin is very similar to the inhibition obtained by preincubation of caldesmon with free Ca++/calmodulin. This result suggests that there is only one Ca++/calmodulin binding domain on caldesmon and this domain appears to be very close to the C21 epitope. Apparently, the Ca++/calmodulin‐binding domain and the actin‐binding domain are very close to each other and may interfere with each other. In an accompanying paper, we have further demonstrated that microinjection of C21 antibody into living chicken embryo fibroblasts inhibit intracellular granule movement, suggesting an in vivo interference with the functional domains [Hegmann et al., 1991: Cell Motil. Cytoskeleton 20:109–120].
Post‐translational incorporation of actin into myofibrils in vitro: Evidence for isoform specificity
Authors: Peng Isaac , Fischman Donald A.
Published: February 2005

The incorporation of actin into myofibrils has been examined in a cell‐free system [Bouché et also Journal of Cell Biology 107:587–596, 1988; Goldfine et all Cellular and Molecular Biology of Muscle Development, 1989]. Actin was translated in a reticulocyte lysate in the presence of 35S‐methionine (35S‐actin) or purified from muscle and labeled with fluorescein‐5‐isothiocyanate (FITC‐actin). Myofibrils were incubated with either 35S‐actin or FITC‐actin and then analyzed by gel electrophoresis or fluorescence microscopy. When myofibrils were incubated with FITC‐actin monomer in the reticulocyte lysate buffer, strong fluorescent labeling was observed in Z‐band regions and less so in I‐bands. No fluorescence was detected in non‐overlap regions of A‐bands. Confocal microscopic analysis of these myofibrils indicated that FITC‐actin was distributed evenly across the diameter of the myofibrils. These observations suggest that actin incorporation in the reticulocyte lysate buffer occurred at sites in the sarcomere which contain actin. In contrast, FITC‐actin showed a variety of non‐physiological incorporation patterns when incubated with myofibrils in the presence of an isotonic buffer (I‐buffer). However, when ATP was added to I‐buffer, FITC‐actin showed a pattern of incorporation into myofibrils similar to that seen in the reticulocyte lysate buffer. Immunoblots indicated that actin of native size was released from myofibrils during incubation in the reticulocyte lysate buffer. No actin release was detected when the myofibrils were incubated in I‐buffer lacking ATP. We used this system to compare the incorporation of actin isoforms into myofibrils. Both α‐ and β‐actins exhibited incorporation into the myofibrils but there was a three‐fold greater incorporation of the α isoform. We propose that the differential affinities of actin isoforms for myofibrils and other cytoskeletal structures could provide a mechanism for actin isoform targeting within the cytoplasm.
Analysis of cDNA clones for Acanthamoeba profilin‐I and profilin‐II shows end to end homology with vertebrate profilins and a small family of profilin genes
Authors: Pollard Thomas D. , Rimm David L.
Published: February 2005

We have cloned and sequenced full length cDNAs for Acanthamoeba profilin‐I and profilin‐II. The genes and the encoded proteins are nearly identical except for the region between bp 121 and 210 where 35% of the nucleotides and 47% of amino acids differ. Most of these substitutions are conservative, although three of them are responsible for the differences in the isoelectric points of the isoforms [Kaiser et al., Cell Biol., 102:221–226, 1986]. The DNA sequence revealed six corrections in the previously published protein sequence of profilin‐I [Ampe et al., J. Biol. Chem. 260:834–840, 1985] and for the first time resolved the ambiguities at the five positions where profilin‐IA and ‐IB differ. The DNA sequence of profilin‐II also allowed us to make two corrections in the protein sequence [Ampeet al., FEBS Lett. 228:17–21, 1988a]. Probes prepared from the cDNAs revealed 1 profilin‐IA gene. one strongly cross‐hybridizing profilin‐I gene and one strongly reacting profilin‐II gene on Southern blots of Acanthamoeba DNA. Weaker reactions with other genomic DNA fragments leave open the possibility of one additional gene each for profilin‐I and profilin‐II. Four different profilin RNAs were resolved on Northern blots. It possible to align the sequences of the three Acanthamoeba profilins with the sequences of nine other profilins from five different phyla. There are only two invariant residues in these profilin sequences, but many pairwise identities and conservative substitutions that indicate considerable divergence of this family of proteins from its ancestral precursor.
In vitro migration of Hydra nematocytes: The influence of the natural extracellular matrix (the mesoglea), of collagen type IV and type I, laminin, and fibronectin on cell attachment, migration parameters, and on patterns of cytoskeletal proteins
Authors: Agosti Charo González , Stidwill Robert P.
Published: February 2005

We have established an in vitro migration system for nematocytes of the fresh water cnidarian Hydra. Nematocytes display a migratory behavior on isolated sheets of the naturally occurring extracellular matrix, the mesoglea, as well as on surfaces coated with collagen type IV or laminin. Cell behavior was analyzed using video microscopic techniques. Average migration speeds of nematocytes on the mesoglea (140 μm/hr) were lower than values reported from in vivo studies (500 μm/hr). Cells on collagen IV moved at about the same average speed (115 μm/hr) as nematocytes on the natural extracellular matrix; those on laminin were considerably slower (20 μm/hr). Attachment but no movement of cells was found on glass or on surfaces coated with collagen type I and fibronectin. In addition to the differential migration speeds, nematocytes displayed distinct morphologies depending on the substratum. In order to elucidate the causes of the observed cell shape and behavior modulations induced by the offered substratum, the arrangement of major cytoskeletal proteins in Hydra nematocytes during the in vitro migration or attachment was investigated. The pattern of F‐actin, myosin, and tubulin was determined by immunocytochemical techniques and confocal laser scanning microscopy in nematocytes moving on the mesoglea, on collagen IV, and on laminin, or in cells attaching to fibronectin. We found that the distribution of the cytoskeletal proteins was strikingly different in moving and in stationary cells. The patterns of cytoskeletal proteins in all nematocytes moving on the different substrata, however, was quite similar.
Compartmentalization and actin binding properties of ABP‐50: The elongation factor‐1 alpha of Dictyostelium
Authors: Dharmawardhane S. , Demma M. , Yang F. , Condeelis J.
Published: February 2005

ABP‐50 is the elongation factor‐1 alpha (EF‐1 alpha) of Dictyostelium discoideum (Yang et al.: Nature 347:494–496, 1990). ABP‐50 is also an actin filament binding and bundling protein (Demma et al.: J. Biol. Chem. 265:2286–2291, 1990). In the present study we have investigated the compartmentalization of ABP‐50 in both resting and stimulated cells. Immunofluorescence microscopy shows that in addition to being colocalized with F‐actin in surface extensions in unstimulated cells, ABP‐50 exhibits a diffuse distribution throughout the cytosol. Upon addition of cAMP, a chemoattractant, ABP‐50 becomes localized in the filopodia that are extended as a response to stimulation. Quantification of ABP‐50 in Triton‐insoluble and‐soluble fractions of resting cells indicates that 10% of the total ABP‐50 is recovered in the Triton cytoskeleton, while the remainder is in the soluble cytosolic fraction. Stimulation with cAMP increases the incorporation of ABP‐50 into the Triton cytoskeleton. The peak of incorporation of ABP‐50 at 90 sec is concomitant with filopod extension. Immunoprecipitation of the cytosolic ABP‐50 from unstimulated cells using affinity‐purified polyclonal anti ABP‐50 results in the coprecipitation of non‐filamentous actin with ABP‐50. Purified ABP‐50 binds to G‐actin with a Kd of approximately 0.09 μM. The interaction between ABP‐50 and G‐actin is inhibited by GTP but not by GDP, while the bundling of F‐actin by ABP‐50 is unaffected by guanine nucleotides. We conclude that a significant amount of ABP‐50 is bound to either G‐ or F‐actin in vivo and that the interaction between ABP‐50 and F‐actin in the cytoskeleton is regulated by cheniotactic stimulation.
Disruption of the golgi apparatus with brefeldin a does not destabilize the associated detyrosinated microtubule network
Authors: Burgess Teresa L. , Skoufias Dimitrios A. , Wilson Leslie
Published: February 2005

Stable subsets of microtubules (MTs) are often enriched in detyrosinated α‐tubulin. Recently it has been found that the Golgi apparatus is associated with a subset of relatively stable MTs and that detyrosinated MTs colocalize spatially and temporally with the Golgi apparatus in several cell lines. To determine whether the Golgi apparatus actively stabilizes associated MTs and thus allows their time‐dependent detyrosination, we have used the drug brefeldin A (BFA) to disrupt the Golgi apparatus and have monitored changes in the Golgi apparatus and MT populations using simultaneous immunofluorescence and fluorescent lectin microscopy. We found that although BFA caused the Golgi apparatus to completely redistribute to the endoplasmic reticulum (ER), the detyrosinated MTs were not disrupted and remained in a juxtanuclear region. By Western blot analysis we found that even after 6 h of continuous exposure of cells to BFA, there was no detectable reduction in the level of detyrosinated α‐tubulin. Simultaneous treatment with nocodazole and BFA led to a complete disruption of all MTs and normal Golgi structure/organization. Upon removal of nocodazole in the continued presence of BFA, we found that the detyrosinated MTs reformed in a compact juxtanuclear location in the absence of an intact Golgi complex. Finally, we found that the detyrosinated MTs colocalized precisely with a BFA‐resistant structure that binds to the lectin, wheat germ agglutinin. We conclude that the juxtanuclear detyrosinated MTs are not actively stabilized by association with BFA‐sensitive Golgi membranes. However, another closely associated structure which binds wheat germ agglutinin may serve to stabilize the juxtanuclear MTs. Alternatively, the MT organizing center (MTOC) and/or MT‐associated proteins (MAPs) may organize and stabilize the juxtanuclear detyrosinated MTs.
The calcium‐induced curvature reversal of rat sperm is potentiated by cAMP and inhibited by anti‐calmodulin
Authors: Lindemann Charles B. , Gardner Tressa K. , Westbrook Evonne , Kanous Kathleen S.
Published: February 2005

Rat sperm, demembranated with 0.1% Triton X‐100, were used to explore the reversal in flagellar curvature induced by calcium ion. As reported earlier (Lindemann and Goltz, Cell Motil. Cytoskeleton, 10:420–431, 1988), the radius of curvature of the flagellar midpiece of rat sperm is controlled by the free Ca2+ concentration. A reversal of the direction of curvature (judged by the asymmetric sperm head) takes place at ≈ 2.5 + 10−6 M free Ca2+.

In our current study, the time course of the curvature change, after elevating free Ca2+ to 3.5 ± 10−4 M, was utilized to assess the effects of the cAMP‐kinase A pathway on the calcium response. In addition, calmodulin's involvement in this response was explored using anti‐calmodulin and Cd2+. The activity state of the sperm models (which could be directly influenced through cAMP) was found to control the rate of curvature change in response to increased free Ca2+. In the most extreme case, fully quiescent sperm did not respond to Ca2+ at all, and cAMP‐primed sperm models completed the response to Ca2+ in two minutes or less.

Anti‐calmodulin demonstrated strong inhibitory effects on the curvature reversal. Cadmium ion was also extremely potent at blocking the response to Ca2+, completely eliminating the curvature reversal at 2 × 10−10 M free Cd2+.

Based on these findings, it appears that the Ca2+‐activated curvature reversal of rat sperm is potentiated by cAMP‐dependent kinase and may be mediated through calmodulin.
Mechanical perturbation of webbed edges in 3T3 cells
Authors: Zand Martin S. , Albrecht‐Buehler Guenter
Published: February 2005

We have previously described actin edge‐bundles (AEBs) as cables of microfil‐aments lining the webbed edges of 3T3 cells (Zand and Albrecht‐Buehler: Cell Motil. Cytoskeleton 13:195–211, 1989). We have suggested that AEBs, along with their cell‐substratum adhesions, resist cortical tension and prevent the collapse of cytoplasm towards the nucleus. In this paper, we report several stages of AEB disassembly and re‐formation induced by the following micro‐manipulations

Scoring of the webbed edge of a 3T3 cells with a microneedle. As a result the sides of the score retracted and the severed AEB appeared to disassemble down to its terminal adhesion points. The retraction stopped after 20–40 seconds and the cells formed a webbed edge with large curvature. Over a period of 20–80 minutes, the new web decreased in length and depth, until it regained its approximate original shape.

Bending of cell processes at acute angles. As a result the processes moved until they projected at right angles to the side of the cell and formed new webs gradually expanded their area. In both cases, the nascent webs were lined by actin edge‐bundles.
Evidence for a gelsolin‐rich, labile F‐actin pool in human polymorphonuclear leukocytes
Authors: Watts Raymond G. , Howard Thomas H.
Published: February 2005

Filamentous (F) actin is a major cytoskeletal element in polymorphonuclear leukocytes (PMNs) and other non‐muscle cells. Exposure of PMNs to agonists causes polymerization of monomeric (G) actin to F‐actin and activates motile responses. In vitro, all purified F‐actin is identical. However, in vivo, the presence of multiple, diverse actin regulatory and binding proteins suggests that all F‐actin within cells may not be identical. Typically, F‐actin in cells is measured by either NBDphallacidin binding or as cytoskeletal associated actin in Triton‐extracted cells. To determine whether the two measures of F‐actin in PMNs, NBDphallacidin binding and cytoskeletal associated actin, are equivalent, a qualitative and quantitative comparison of the F‐actin in basal, non‐adherent endo‐toxin‐free PMNs measured by both techniques was performed. F‐actin as NBD‐phallacidin binding and cytoskeletal associated actin was measured in cells fixed with formaldehyde prior to cell lysis and fluorescent staining (PreFix), or in cells lysed with Triton prior to fixation (PostFix). By both techniques, F‐actin in PreFix cells is higher than in PostFix cells (54.25 ± 3.77 vs. 23.5 ± 3.7 measured as mean fluorescent channel by NBDphallacidin binding and 70.3 ± 3.5% vs. 47.2 ± 3.6% of total cellular actin measured as cytoskeletal associated actin). These results show that in PMNs, Triton exposure releases a labile F‐actin pool from basal cells while a stable F‐actin pool is resistant to Triton exposure. Further characterizations of the distinct labile and stable F‐actin pools utilizing NBDphallacidin binding, ultracentrifugation, and electron microscopy demonstrate the actin released with the labile pool is lost as filament. The subcellular localization of F‐actin in the two pools is documented by fluorescent microscopy, while the distribution of the actin regulatory protein gelsolin is characterized by immunoblots with antigelsolin. Our studies show that at least two distinct F‐actin pools coexist in endotoxin‐free, basal PMNs in suspension: (1) a stable F‐actin pool which is a minority of total cellular F‐actin, Triton insoluble, resistant to depolymerization at 4°C, gelsolin‐poor, and localized to submembranous areas of the cell; and (2) a labile F‐actin pool which is the majority of total cellular F‐actin, Triton soluble, depolymerizes at 4°C, is gelsolin‐rich, and distributed diffusely throughout the cell. The results suggest that the two pools may subserve unique cytoskeletal functions within PMNs, and should be carefully considered in efforts to elucidate the mechanisms which regulate actin polymerization and depolymerization in non‐muscle cells.
Increased calmodulin affects cell morphology and mRNA levels of cytoskeletal protein genes
Authors: Rasmussen Colin D. , Means Anthony R.
Published: February 2005

We have previously described stable mouse C127 cell lines in which a CaM mini‐gene has been expressed in a bovine papilloma virus‐based expression vector (Rasmussen and Means: EMBO J. 6:3961–3968. 1987). Elevation of CaM to levels five‐fold higher than in control cells caused an acceleration in cell cycle progression by reducing the length of the G1 period. When these cell lines were originally isolated it was observed that cells in which CaM levels were increased had a flattened morphology. In this study we have examined the localization of actin, vimentin, and tubulin in these cells as compared to the BPV‐transformed control cell line in order to determine if changes in shape were accompanied by differences in the cytoskeletal organization. Cell‐cycle‐dependent changes in the levels of mRNAs for histone H4, glyceraldehyde‐3‐phosphate dehydrogenase, β‐actin, vimentin, and β‐tubulin have also been examined. Our results indicate that increased CaM causes differences in the organization of microfilaments, intermediate filaments, and microtubules and that these changes are accompanied by selective differences in the cell‐cycle‐dependent expression of some mRNAs. Elevated CaM was also correlated with a reduced stability of β‐tubulin mRNA. These studies indicate that CaM has pleiotropic effects on cell function and suggest that stable cell lines with altered CaM levels may provide a useful model system for understanding the moiecular basis of CaM‐dependent regulation of cellular processes.
Trout sperm swimming patterns and role of intracellular Ca++
Authors: Boitano Scott , Omoto Charlotte K.
Published: February 2005

We investigated the swimming patterns of trout sperm using computer‐assisted analyses of video microscopy. Under full activation conditions, in which 80–100% of sperm activate their motility, sperm swim in circular paths for 2–5 sec, followed by 30–60 sec of a more linear swimming, and, finally, cessation of movement, with a straightening of the flagella. Threshold activation, in which 50% of the sperm activate, is characterized by circular patterns of swimming for less than 20 sec, with straightened flagella upon cessation. Full activation and threshold activation are observed in low‐K+ solution or in an Mg++ ‐supplemented K+ solution. Similarities in swimming patterns in low‐K+ solution and in a Mg++ ‐supplemented K+ solution suggest a common underlying mechanism of activation. Initiation of movement in solutions with high Ca++ to K+ ratio is similar to activation in K+ ‐free solution. However, sperm in Ca++ ‐supplemented media resume circular swimming within 20–25 sec after activation, and, upon cessation of movement, the flagella are frequently cane shaped or bent. Differences in swimming patterns upon activation by high Ca++ concentration suggest additional effects of Ca++ on regulating swimming patterns. We used the fluorescent Ca++ indicator Fluo‐3 to measure changes in intracellular Ca++ concentration upon activation. Intracellular Ca++ concentration transiently increases upon activation, with peak Ca++ concentration coinciding with the period of circular swimming. This transient increase in Ca++ concentration is seen in the absence of external Ca++, providing strong evidence for the released of Ca++ from intracellular stores upon activation.
Reactivation of newt lung cilia: Evidence for a possible temperature‐ and MgATP‐induced activation mechanism
Authors: Hard Robert , Cypher Christopher
Published: February 2005

Optimal conditions have been developed for the isolation and reactivation of highly coupled, demembranated ciliary axonemes from newt lungs [Hard, Cypher, and Schabtach, 1988, Cell Motil. Cytoskeleton 10:271–284]. In the present study, the motility of these cilia was further characterized by examining the effects of nucleotides, divalent cations, and temperature on beat frequency. When exposed to a reactivating solution containing Mg2+ and ATP, nearly 100% of the axonemes were motile and beat at frequencies of 0–50 Hz, depending on [MgATP] and temperature. Divalent cations were required for movement, with Mg2+ 2–3 times more effective than Ca2+. There was no absolute requirement for Ca2+ for motility. The beat frequencies obtained with fixed ATP and varying Mg2+ concentrations indicate that MgATP serves as the actual substrate. The effects of MgATP on beat frequency depended on the degree of mechanochemical coupling and temperature and MgATP‐induced transition between two distinct states whose maximum beat frequencies differ by 200–300%.
Colchicine‐binding sites of brain tubulins from an Antarctic fish and from a mammal are functionally similar, but not identical: Implications for microtubule assembly at low temperature
Authors: Skoufias Dimitrios A. , Wilson Leslie , Detrich H. William
Published: February 2005

The tubulins of Antarctic fishes possess adaptations that favor microtubule for mation at low body temperatures (Detrich et al.: Biochemistry 28:10085–10093, 1989). To determine whether some of these adaptations may be present in a domain of tubulin that participates directly or indirectly in lateral contact between microtubule protofilaments, we have examined the energetics of the binding of colchicine, a drug thought to bind to such a site, to pure brain tubulins from an Antarctic fish (Notothenia gibberifrons) and from a mammal (the cow, Bos taurus), At temperatures between 0 and 200C, the affinity constants for colchicine binding to the fish tubulin were slightly smaller (1.5–2.6‐fold) than those for bovine tubulin. van't Hoff analysis showed that the standard enthalpy changes for colchicine binding to the two tubulins were comparable (δH° = + 10.6 and + 7.4 kcal mol‐1 for piscine and bovine tubulins, respectively), as were the standard entropy changes (δS° = +61.3 eu for N. gibberifrons tubulin, +51.2 eu for bovine tubulin). At saturating concentrations of the ligand, the maximal binding stoichiometry for each tubulin was ∼ 1 mol colchicine/mol tubulin dimer. The data indicate that the colchicine‐binding sites of the two tubulins are similar, but probably not identical, in structure. The apparent absence of major structural modifications at the colchicine site suggests that this region of tubulin is not involved in functional adaptation for low‐temperature polymerization. Rather, the colchicine site of tubulin may have been conserved evolutionarily to serve in vivo as a receptor for endogenous molecules (i.e., “colchicine‐like” molecules or MAPs) that regulate microtubule assembly.
Human cardiac and skeletal muscle spectrins: Differential expression and localization
Authors: Vybiral Tomas , Winkelmann John C. , Roberts Robert , Joe Eun‐Hye , Casey Douglas L. , Williams Joseph K. , Epstein Henry F.
Published: February 2005

We describe multiple human cardiac and skeletal muscle spectrin isoforms. Cardiac muscle expresses five erythroid α,β spectrin‐reactive isoforms with estimated MR's of 280, 274, 270, 255, and 246 kD, respectively At least one nonerythroid α‐spectrin of MR 284 kD is expressed in heart. While skeletal muscle shares the 280, 270, and 246 kD erythroid spectrins, it expresses an immunologically distinct 284 kD nonerythroid α‐spectrin isoform. The 255 kD erythroid β‐spectrin isoform is specific for cardiac tissue. By immunocytochemistry, both erythroid β‐ and nonerythroid α‐spectrins are localized to costameres, the plasma membrane, and the neuromuscular junctional region.
Different assembly properties of cod, bovine, and rat brain microtubules
Authors: Fridén Bo , Stromberg Elisabeth , Wallin Margareta
Published: February 2005

Assembly properties of cod, bovine, and rat brain microtubules were compared. Estramustine phosphate, heparin, poly‐L‐aspartic acid, as well as NaCl, inhibited the assembly and disassembled both bovine and rat microtubules by inhibition of the binding between tubulin and MAPs. The assembly of cod brain microtubules was in contrast only marginally affected by these agents, in spite of a release of the MAPs. The results suggest that cod tubulin has a high intrinsic ability to assemble. This was confirmed by studies on phosphocellulose‐purified cod tubulin, since the critical concentration for assembly was independent of the presence or absence of MAPs. The results show therefore that cod brain tubulin has, in contrast to bovine and rat brain tubulins, a high propensity to assemble under conditions which normally require the presence of MAPs.

Even if cod MAPs, which have an unusual protein composition, were not needed for the assembly of cod microtubules, they were able to induce assembly of bovine brain tubulin. Both cod and bovine MAPs bound to cod microtubules, and bovine MAPI and MAP2 bound to, and substituted at least the 400 kDa cod protein. This suggests that the tubulin‐binding sites and the assembly‐stimulatory ability of MAPs are common properties of MAPs from different species, independent of the tubulin assembly propensity.
Heterogeneity of microtubule organizing center components as revealed by monoclonal antibodies to mammalian centrosomes and to nucleus‐associated bodies from Dictyostelium
Authors: Sellitto Caterina , Kimble Mary , Kuriyama Ryoko
Published: February 2005

The molecular composition of two morphologically distinct microtubule‐organizing centers (MTOCs) was compared by probing with monoclonal antibodies raised against (i) nucleus‐associated bodies (NABs) isolated in a complex with nuclei from the cellular slime mold Dictyostelium discoideum and (ii) mammalian mitotic spindles isolated from Chinese hamster ovary (CHO) cells. The staining patterns observed by immunofluorescence microscopy in whole CHO cells and Dictyostelium amoebae showed that the distribution of thirteen MTOC antigens is heterogeneous. Not all antibodies recognized the MTOC in both interphase and mitosis. Most of the anti‐MTOC antibodies cross‐reacted with other cellular organelles such as nuclei, Golgi apparatus‐like aggregates and cytoskeletal elements. Two antibodies, CHO3 and AX3, recognized phosphorylated epitopes present in both mammalian centrosomes and Dictyostelium NABs. On immunoblots, most of the antibodies showed multiple bands, often of high molecular weight, indicating that the antigenic determinants are shared among different molecules. One antibody inhibited the regrowth of microtubules onto centrosomes in vitro after addition of exogenous tubulin to detergent‐lysed CHO cells on coverslips; this antibody binds to an antigen(s) that might be essential for the microtubule‐nucleating activity of centrosomes. These observations demonstrate that molecular components in different MTOCs exhibit a variety of distinct subcellular localizations and functional properties, and that some antigenic molecules have been conserved among morphologically distinct MTOCs. © 1992 Wiley‐Liss, Inc.
An immunofluorescence study of the effects of ultraviolet radiation on the organization of microfilaments, keratin intermediate filaments, and microtubules in human keratinocytes
Authors: Zamansky Glen B. , Nguyen Uyensa , Chou Lih‐Nan
Published: February 2005

Indirect immunofluorescence microscopy has been used to investigate the ultraviolet (UV) radiation induced disruption of the organization of microfilaments, keratin intermediate filaments, and microtubules in cultured human epidermal keratinocytes. Following irradiation, concurrent changes in the organization of the three major cytoskeletal components were observed in cells incubated under low Ca2+ (0.15 mM) conditions. UV irradiation induced a dose‐dependent condensation of keratin filaments into the perinuclear region. This collapse of the keratin network was accompanied by the reorganization of microfilaments into rings and a restricted distribution of microtubules, responses normally elicited by exposure to high Ca2+ (1.05 mM) medium. The UV induced alteration of the keratin network appears to disrupt the interactions between keratin and actin, permitting the reorganization of actin filaments in the absence of Ca2+ stimulation.

In addition to the perinuclear condensation of keratin filaments, UV irradiation inhibits the Ca2+ induced formation of keratin alignments at the membrane of apposed cells if UV treatment precedes exposure to high Ca2+ medium. Incubation of keratinocytes in high Ca2+ medium for 24 hours prior to irradiation results in the stabilization of membrane associated keratin alignments and a reduced susceptibility of cytoplasmic keratin filaments to UV induced disruption. Unlike results from investigations with isogenic skin fibroblasts, no UV induced disassembly of microtubules was discernible in irradiated human keratinocytes. © 1992 Wiley‐Liss, Inc.
Evolution of the flagellar waveform of ram spermatozoa in relation to the degree of epididymal maturation
Authors: Chevrier C. , Dacheux J.‐L.
Published: February 2005

Motility and flagellar movement of ram spermatozoa along the epididymis were analysed in vitro. From the caput to the cauda of the epididymis, the percentage of motile and progressive spermatozoa increases. No flagellar bending was observed in spermatozoa from the testis or the epididymal anterior caput. When spermatozoa reached the distal caput of the epididymis, a static curvature, associated with an initiation of the flagellar beating, appeared on the flagella. This curvature normally disappeared during epididymal transit. Its disappearance was associated with an increase in the flagellar beat efficiency. Our results suggest that the initiation of motility is related to two mechanisms involving: (1) the presence of a transient static curvature, and (2) the establishment of a symmetric regular beating of the flagellum. © 1992 Wiley‐Liss, Inc.
Timing of mitotic chromosome loss caused by the ncd mutation of Drosophila melanogaster
Authors: Nelson Catherine R. , Szauter Paul
Published: February 2005

We studied the timing of mitotic loss of maternally and paternally derived chromosomes among the progeny of Drosophila melanogaster females homozygous for an amorphic mutation in ncd, a gene encoding a kinesin‐like protein. In order to determine the division at which chromosome loss occurs, we estimated the fraction of XO nuclei resulting from X chromosome loss by scoring the phenotype of 47 adult cuticular landmarks in 160 XX–XO mosaics (gynandromorphs) derived from maternal X chromosome loss, and 33 gynandromorphs derived from paternal X chromosome loss. The results show that while most of the mitotic loss of maternally derived chromosomes occurs at the first cleavage division, the mitotic loss of paternally derived chromosomes occurs only at the second and later divisions. This means that paternally derived chromosomes are immune from the effects of ncd prior to karyogamy, which occurs after the first cleavage division. We discuss the implications of these results for the function of the ncd gene product and for other kinesin‐like proteins in Drosophila. © 1992 Wiley‐Liss, Inc.
Activation of maternal centrosomes in unfertilized sea urchin eggs
Authors: Schatten Heide , Walter Marika , Biessmann Harald , Schatten Gerald
Published: February 2005

Centrosomes are undetectable in unfertilized sea urchin eggs, and normally the sperm introduces the cell's microtubule‐organizing center (MTOC) at fertilization. However, artificial activation or parthenogenesis triggers microtubule assembly in the unfertilized egg, and this study explores the reappearance and behavior of the maternal centrosome. During activation with A23187 or ammonia, microtubules appear first at the cortex; centrosomal antigen is detected diffusely throughout the entire cytoplasm. Later, the centrosome becomes more distinct and organizes a radial microtubule shell, and eventually a compact centrosome at the egg center organizes a monaster. In these activated eggs, centrosomes undergo cycles of compaction and decompaction in synchrony with the chromatin, which also undergoes cycles of condensation and decondensation. Parthenogenetic activation with heavy water (50% D2O) or the microtubule‐stabilizing drug taxol (10 μM) induces numerous centrosomal foci in the unfertilized sea urchin egg. Within 15 min after incubation in D2O, numerous fine centrosomal foci are detected, and they organize a connected network of numerous asters which fill the entire egg. Taxol induces over 100 centrosomal foci by 15 min after treatment, which organize a corresponding number of asters. The centrosomal material in either D2O‐ or taxol‐treated eggs aggregates with time to form fewer but denser foci, resulting in fewer and larger asters. Fertilization of eggs pretreated with either D2O or taxol shows that the paternal centrosome is dominant over the maternal centrosome. The centrosomal material gradually becomes associated with the enlarged sperm aster. These experiments demonstrate that maternal centrosomal material is present in the unfertilized egg, likely as dispersed undetectable material, which can be activated without paternal contributions. At fertilization, paternal centrosomes become dominant over the maternal centrosomal material. © 1992 Wiley‐Liss, Inc.