bluuebunny/crossref_metadata_2025 huggingface dataset:
Light‐induced stop response in Chlamydomonas reinhardtii: Occurrence and adaptation phenomena
Authors: Hegemann Peter , Bruck Benjamin
Published: February 2005

In darkness Chlamydomonas cells swim forward with a helical motion of low amplitude. When cells are exposed to light conditions they are not adapted to, they perform direction changes or stop responses depending on how far the stimulant irradiance is shifted from the former adaptation level. Here we present the utility of a commercially available motion analysis system for the analysis of the Chlamydomonas stop response.

Chlamydomonas cells stop in darkness only occasionally with a random temporal distribution but with a highly increased frequency after a flash or a step‐up light stimulation. The delay time, tD, defined as the minimal time difference between flash and a light‐induced stop, was below 50 ms. The reaction time, tR, defined as the time difference between flash and the maximal probability for a cell to stop was found to be 140 ms. During a stop the cells swim revers for some 300 ms with 20% of the forward swimming speed.

To a given stimulation program cells adapt with a first‐order kinetic. In the case of a single step‐up or step‐down stimulation this adaptation consists of a single stop response followed by direction changes which decrease in frequency to a certain steady‐state level. To repetitive light pulses the cells respond with a gradual disappearance of step‐up stop responses and a concurrent appearance of step‐down responses.

External calcium influences the stop response in a multifunctional way. For stop responses to occur 300 nM calcium are required. At increasing calcium concentrations the duration of a stop response is extended. Besides light, calcium regulates the time course of light‐adaption and the absolute adaptation level.

A kinetic model for the description of adaptation is presented.
Nucleus‐basal body connector in Chlamydomonas: Evidence for a role in basal body segregation and against essential roles in mitosis or in determining cell polarity
Authors: Wright Robin L. , Adler Sally A. , Spanier Jonathan G. , Jarvik Jonathan W.
Published: February 2005

In the unicellular biflagellate green alga Chlamydomonas reinhardtii each basal body is linked to the nucleus by a fibrous nucleus‐basal body connector (NBBC) that contains the calcium‐binding protein centrin. (Wright et al.: Journal of Cell Biology 101:1903–1912.; Salisbury et al.: Journal of Cell Biology 107:635–642; Huang et al.: Journal of Cell Biology 107:121–131). In order to explore the cellular function of the NBBC we used antiserum directed against centrin to examine a number of mutants known to be defective for basal body assembly and/or localization. Of three variable flagella‐number mutants examined, one, vfl‐2, is dramatically defective with respect to the NBBC in that (1) the union between basal bodies and nucleus is very labile, (2) there is no detectible centrin in the NBBC region, and (3) total cellular centrin levels are reduced 75–80% relative to wild type. The existence of these defects in a mutant incapable of maintaining normal flagellar number supports the view that the NBBC plays an important role in determining proper basal body localization and/or segregation. In contrast to vfl‐2, the mutants vfl‐1, vfl‐3, uni‐1, and bald‐2 contain approximately normal levels of centrin and possess stable NBBCs. The observation of NBBCs in the mutant bald‐2, which lacks all but very rudimentary basal bodies, indicates that the assembly of the NBBC does not require fully formed basal bodies and that such assembly may not require basal bodies at all. Finally, the possibility that the NBBC is required for induction of gene expression following deflagellation was tested by examining vfl‐2 for such induction. Results indicate that the connector does not play a necessary role in the induction process.
Epidermal growth factor induces the accumulation of calpactin II on the cell surface during membrane ruffling
Authors: Campos‐Gonzalez Roberto , Kanemitsu Martha , Boynton Alton L.
Published: February 2005

Confluent and proliferatively quiescent T51B rat liver epithelial cells provide a cellular model for the study of epidermal growth factor (EGF) effects in non‐neoplastic cells. Immunoreactive calpactin II, a well‐known substrate for EGF‐receptor kinase, was found predominantly in the cytosol, although a second im‐munoreactive pool was found in a Triton X‐100‐extractable membrane fraction. Stimulation with EGF resulted in a rapid and transient (2–;5 min) formation of ruffles at the cell surface and at the cell‐cell contacts. Both calpactin II and filamentous actin were found co‐localized at the membrane ruffles. Immunopre‐cipitations of membrane‐bound calpactin II from 32P‐labeled cells indicate a transient EGF‐dependent phosphorylation of calpactin II correlating with membrane ruffling. These results suggest a temporal (2–5 min) function for calpactin II at the plasma membrane during the EGF‐induced mitogenesis of T51B cells.
Purification and characterization of an 85 kDa talin‐binding fragment of vinculin
Authors: Groesch Mary E. , Otto Joann J.
Published: February 2005

Vinculin and talin are adhesion plaque proteins which have been shown to interact with each other in vitro. In order to begin to investigate where the talin‐binding domain is in vinculin, vinculin was digested with Staphylococcus aureus V8 protease to generate two major fragments of 85 and 30 kDa, and these fragments were purified. Nitrocellulose overlays with 125I‐talin and the 125I‐85 kDa vinculin fragment and sucrose density gradient centrifugation demonstrated that the talin‐binding domain was localized to the 85 kDa vinculin fragment. Quantification of 125I‐talin binding in the overlays showed that four times more talin bound to the 85 kDa fragment as compared to intact vinculin. Competitive immunoprecipitation experiments demonstrated that unlabeled 85 kDa fragment, was about three fold more effective at competing for 125I‐85 kDa binding to talin than was unlabeled vinculin. These results suggest that the 30 kDa fragment inhibits the vinculin‐talin interaction even though the talin‐binding domain is localized in the 85 kDa fragment.
Reorganization of cortical actin microfilaments and microtubules at preprophase and mitosis in wheat root‐tip cells: A double label immunofluorescence study
Authors: McCurdy David W. , Gunning Brian E. S.
Published: February 2005

Following our recent description [McCurdy et al.: Protoplasma. 147:204–206, 1988] of arrays of transverse cortical microfilaments (MFs) in preprophase roottip cells of wheat (Triticum aestivum L. cv. Kite), we have performed double label immunofluorescence microscopy to correlate the formation of these arrays with the known rearrangement of cortical microtubules (MTs) that occurs during preprophase. At early preprophase, indicated by a broad (i.e., young) preprophase band (PPB) of MTs, actin MFs are transverse only in the central region of the cell cortex. By late preprophase, however, cells that possess a mature (i.e., narrow) PPB of MTs have arrays of transverse MFs that occupy the entire cortical surface of the cell. Thus, apart from the PPB zone, the transverse MFs in these arrays do not colocalize with transverse cortical MTs. Depolymerization of MTs using the herbicide oryzalin does not effect the arrays of cortical MFs; however, experiments using cytochalasin B in combination with oryzalin indicate that cellular MTs are necessary for the formation of the arrays of transverse cortical MFs. The arrays of cortical MFs disintegrate during prophase into short fragments of random, filamentous actin. This situation persists until the completion of cytokinesis. The absence of MFs during mitosis in densely‐cytoplasmic meristematic cells of wheat root tips indicates that filamentous actin may not have a universal function in plant cell division. The possible function of the arrays of cortical MFs in preprophase cells is discussed.
Effects of brain microtubule‐associated proteins on microtubule dynamics and the nucleating activity of centrosomes
Authors: Bré M. H. , Karsenti E.
Published: February 2005

In this paper, we report on the effect of brain microtubule‐associated proteins (MAPs) on the dynamic instability of microtubules as well as on the nucleation activity of purified centrosomes. Under our experimental conditions, tau and MAP2 have similar effects on microtubule nucleation and dynamic instability. Tau increases the apparent elongation rate of microtubules in proportion to its molar ratio to tubulin, and we present evidence indicating that this is due to a reduction of microtubule instability rather than to an increase of the on rate of tubulin subunits at the end of growing microtubules. Increasing the molar ratio of tau over tubulin leads also to an increase in the average number of microtubules nucleated percentrosome. This number remains constant with time. This suggests that the number of centrosome‐nucleated microtubules at steady state can be determined by factors that are not necessarily irreversibly bound to centrosomes but, rather, affect the dynamic properties of microtubules.
Partial characterization of a carotenoid droplet ATPase and its possible significance in carotenoid droplet dispersion in goldfish xanthophores
Authors: Wu Bei‐yue , Yu Fu‐xin , Lynch Thomas J. , Taylor John D. , Tchen T. T.
Published: February 2005

The dispersion of carotenoid droplets in permeabilized goldfish xanthophores is dependent on ATP, F‐actin, and cytosol. We report here that the motor (ATPase, translocator) resides with the permeabilized cell remnants and not in the cytosol. We also report that the carotenoid droplets have an ATPase that is not conventional myosin, dynein, or an ion pump. Its activity appears to correlate with the actin content of the carotenoid droplet preparation. A carotenoid droplet protein of Mr 72,000 (p72) is shown to be labeled by irradiation with 8‐azido‐ATP with concomitant loss of ATPase activity of the carotenoid droplets. We propose that this protein may be the ATPase responsible for carotenoid droplet dispersion.
A 70 kD microtubule‐binding protein from starfish eggs: Purification, characterization, and localization during meiosis and mitosis
Authors: Hosoya Natsumi , Hosoya Hiroshi , Mohri Toshiko , Mohri Hideo
Published: February 2005

A microtubule‐binding protein was purified from eggs of the starfish, Asterias amurensis, through several steps of purification including the taxol‐dependent procedure [Vallee, 1982, J. Cell Biol. 92:435–442]. This protein consists of a single polypeptide chain having an apparent molecular mass of 70 kD determined by SDS‐PAGE. The 70 kD protein was identified as a unique microtubulebinding protein, judging from electrophoretic mobility, cleavage pattern by limited proteolysis, heat stability, and immunocrossreactivity. The 70 kD protein binds to brain and egg microtubules. It does not promote assembly of brain tubulin, but promotes that of egg tubulin in vitro in a concentration‐dependent manner.

Using indirect immunofluorescence and immunoelectron microscopy with the anti‐70 kD protein antibody, we analyzed the cellular localization of the 70 kD protein in starfish oocytes and eggs during both meiotic maturation (meicsis) and first cleavage (mitosis). Immunofluorescence studies showed that the 70 kD protein localized on microtubule structures spread widely throughout the cytoplasm, the sperm aster, and the microtubules making up the mitotic apparatus through both meiosis and mitosis. The antibody, however, did not recognize sperm axonemes. These results were confirmed by immunoelectron microscopy. Using a colloidal gold technique, the 70 kD protein was localized along the microtubules in vivo.

This 70 kD protein is the first microtubule‐binding protein that has been shown to localize along the microtubules in oocytes and eggs throughout meiosis and mitosis and to promote microtubule assembly. The 70 kD protein may be involved in the dynamic changes of microtubule structures occurring within oocytes and eggs.
The centrosomal cycle: Visualization in PtK cells by a monoclonal antibody to a centrosomal 32 kd protein
Authors: Joswig Gaby , Petzelt Christian
Published: February 2005

A monoclonal antibody prepared against a crude centrosome fraction from PtK cells reacts exclusively with centrosomes. Using Western blotting techniques, the antigen was identified as a protein with a molecular weight of 32 kd. Using this probe it is possible to follow pronounced shape changes of the centrosome through the cell cycle and to study its replication. When the microtubular cytoskeleton is removed by nocodazole, neither the shape nor the three‐dimensional organization of the centrosome inside the cell are altered. Moreover, in spite of the cell cycle arrest caused by nocodazole, the centrosomal cycle proceeds, thus indicating its independence from the intact cytoskeleton and supporting its role as a semiautonomous organelle. On the basis of these results we hypothesize that the centrosome has two distinct functions: in the non‐growing compact state during mitosis the centrosome serves as the pole organizer and during interphase it functions as the “cytocenter”.
Nucleotide specificity for the bidirectional transport of membrane‐bounded organelles in isolated axoplasm
Authors: Leopold Philip L. , Snyder Robert , Bloom George S. , Brady Scott T.
Published: February 2005

Video microscopy of isolated axoplasm from the squid giant axon permits correlated quantitative analyses of membrane‐bounded organelle transport both in the intact axoplasm and along individual microtubules. As a result, the effects of experimental manipulations on both anterograde and retrograde movements of membrane‐bounded organelles can be evaluated under nearly physiological conditions. Since anterograde and retrograde fast axonal transport are similar but distinct cellular processes, a systematic biochemical analysis is important for a further understanding of the molecular mechanisms for each. In this series of experiments, we employed isolated axoplasm of the squid to define the nucleoside triphosphate specificity for bidirectional organelle motility in the axon. Perfusion of axoplasm with 2–20 mM ATP preserved optimal vesicle velocities in both the anterograde and retrograde directions. Organelle velocities decreased to <50% of optimal values when the axoplasm was perfused with 10–20 mM UTP, GTP, ITP, or CTP with simultaneous depletion of endogenous ATP with hexokinase. Under the same conditions, TTP and ATP‐γ‐S were unable to support significant levels of transport. None of the NTPs tested had a differential effect on anterograde vs. retrograde movement of vesicles. Surprisingly, several inconsistencies were revealed when a comparison was made between these results and nucleoside triphosphate specificities that have been reported for putative organelle motors by using in vitro assays. These data may be used in conjunction with data from well‐defined in vitro assays to develop models for the molecular mechanisms of axonal transport.
Ultrastructural cytoskeleton alterations and modification of actin expression in the NIH/3T3 cell line after transformation with Ha‐ras‐Activated oncogene
Authors: Lombardi Luciano , Ballinari Dario , Bongarzone Italia , Migliari Marco , Mondellini Piera , Traversai Catia , Modina Silvia
Published: February 2005

Cytoskeleton alterations of NIH/3T3 fibroblast monolayers transfected with Haras‐activated oncogene were studied by immunofluorescence, immunoelectron microscopy, and immunoelectrophoretic analysis of actin isoforms. Transformation foci were found to consist of cells with a round shape and rare stress fibers that spread sparsely, forming rare focal contacts and fibronexuses. The loss of stress fibers in transformed cells was confirmed by staining with rhodamine‐phalloidin and with a fluorescinated anti‐non‐muscle cell actin antibody. The transformed cells were anchored to the substrate prominently by filaments that contained fibronectin, as showed by immunoelectron microscopy. A down‐regulation of α‐actin isoform was observed by immunofluorescence and immunoblotting analysis using a specific monoclonal antibody. The diffuse distribution of α‐actin, lacking a specific association with stress fibers, challenges the hypothesis of a connection between α‐actin down‐regulation and stress fiber loss.
Formation, transport, contraction, and disassembly of stress fibers in fibroblasts
Authors: Giuliano Kenneth A. , Lansing Taylor D.
Published: February 2005

Swiss mouse 3T3 fibroblasts grown on a solid substrate in the presence of 10% serum exhibit cell movement, organelle transport, and cytokinesis. When the serum concentration in the culture medium is decreased to 0.2% for 48 h the serum‐deprived cells virtually stop locomoting, spread, decreased organelle transport, and exhibit extensive arrays of stress fibers that are visible with video‐enhanced differential interference contrast microscopy and that also incorporate fluorescent analogs of actin and conventional myosin (myosin II). The stress fibers form in a constitutive mannet at the cytoplasm‐membrane interface, transport toward the nucleus, and then disappear. The rate of transport of these fibers is quite heterogeneous with average rates in the range of 10–20 μm/h. When serum‐deprived cells are stimulated with mitogens such as 10% serum or 10 nM thrombin, many of the stress fibers immediately begin to shorten, suggesting a contraction. The rate of shortening is approximately two orders of magnitude slower than that of unloaded smooth muscle cells. The fiber shortening is often accompanied by retraction of the edges of the cell and continues for at least the 1st hour post‐stimulation.
Molecular cloning and expression of sea urchin embryonic ciliary dynein β heavy chain
Authors: Foltz Kathleen R. , Asai David J.
Published: February 2005

The determination of the structure and the expression of dynein during embryonic development are central to the understanding of dynein function. As an important first step toward these objectives, cDNAs encoding portions of sea urchin ciliary dynein were identified by antibody screening of a sea urchin cDNA expression library. Bacause of the complete lack of protein sequence data, it was first necessary to prove the identity of the dynein cDNAs. Of the five cDNA inserts initially cloned, one, designated P72A1, was characterized extensively. Four independent criteria demonstrated that P72A1 encoded a portion of a dynein heavy chain. (1) The β‐galactosidase‐P72A1 fusion protein affinity‐purified dynein‐specific antibodies from crude antiserum. (2) Two other antisera to dynein, raised independently of the antiserum used to screen the cDNA library, reacted with the fusion protein. (3) A new antiserum raised against the fusion protein reacted with authentic dynein heavy chain on Western blots and stained embryonic cilia by indirect immunofluorescence microscopy. (4) Two new antisera, elicited against opposite ends of the P72A1 open reading frame, each reacted with authentic dynein heavy chain protein. Western blot analyses of dissociated dynein heavy chains revealed that P72A1 encoded a portion of the β heavy chain. Epitope mapping experiments confirmed the identity of P72A1 as part of the βheavy chain and also demonstrated that P72A1 encoded epitopes of the carboxyl‐terminal fragment B domain of the dynein β heavy chain. Northern blot analyses of poly(A)+ RNA revealed that P72A1 hybridized with a large RNA species ca. 12.5 kb in length. The dynein mRNA concentration increased during embryonic development. Dot blot analyses of RNA isolated at various times after embryo deciliation demonstrated that the dynein β heavy chain mRNA accumulated rapidly in response to deciliation. The accumulation was similar to but not identical with the induction of tubulin mRNA in response to the same stimulus.
Role of the cytoskeleton during injury‐induced cell migration in corneal endothelium
Authors: Gordon Sheldon R. , Staley Carol A.
Published: February 2005

The role of microfilaments and microtubules during injury‐induced cell migration of corneal endothelial cells in situ along their natural basement membrane has been investigated using organ culture. In the noninjured tissue, actin is localized at or near the plasma membrane, whereas tubulin is observed as a delicate lattice pattern throughout the cytoplasm. Twenty‐four hours after a circular freeze injury, cells surrounding the wound area extend processes into this region. Fluorescent microscopy using phallotoxins and anti‐tubulin antibodies demonstrated the presence of stress fibers and microtubule reorganization within these cells. Between 24 and 48 h post‐injury endothelial cells move into the wound region, and by 48 h, the injury zone is repopulated and the monolayer is becoming resstablished. When injured corneas are placed in media containing 5 × 10−7 M cytochalasin B, endothelial cell migration occurs: but it is slow, and wound closure is not complete even by 72 h. In contrast, when tissues are cultured in the presence of 10−8 M colchicine, cell movement is greatly reduced, complete wound closure does not occur, and endothelial cells at the wound edge fail to display extensions typical of migrating cells. Furthermore, when injured endothelia are exposed to 0.05 μg/ml of actinomycin D for 15 min within the first hour after injury and transferred back into culture media lacking the drug for the duration of the experiment, migration does not occur and the wound persists. These actinomycin D treated cells remain viable as shown by their ability to incorporate 3H‐uridine and 3H‐thymidine. Fluorescence microscopy of actinomycin D treated tissues revealed the presence of stress filaments but disorganized microtubule patterns. Interestingly, 24 h after injury, if the tissue is exposed to actinomycin D, even for periods of up to 1 h, migration is not inhibited. Our results indicate that injury‐induced endothelial cell movement appears to be more dependent on microtubule than microfilament reorganization and may require a critical timing of macromolecular synthesis.
Cytoskeletal dynamics in rabbit synovial fibroblasts: I. Effects of acrylamide on intermediate filaments and microfilaments
Authors: Aggeler Judith , Seely Keith
Published: February 2005

Rabbit synovial fibroblasts respond to changes in cell shape and cytoskeletal architecture by altering specific gene expression. We have tested the ability of acrylamide, a neurotoxin that alters the distribution of intermediate filaments in cultured PtKl cells, to induce metalloprotease expression in synovial fibroblasts. Cells treated with 2–20 mM acrylamide for 5 to 24 h underwent shape changes similar to cells treated with the tumor promoter phorbol myristate acetate. Intermediate filaments visualized with anti‐vimentin antibodies did not collapse into a perinuclear cap in these rounded cells, but were still present in the extended cell processes. Unexpectedly, when actin was visualized in acrylamide‐treated cells, extensive dissociation and clumping of microfilaments was observed. Concentrations of acrylamide > 10 mM were cytotoxic, but cells recovered completely after 24 h incubation with 5 mM acrylamide. Like other agents that alter cell shape and actin distribution in synovial fibroblasts, acrylamide also induced expression of the secreted metalloprotease collagenase. Although some recent evidence suggests that acrylamide may be able to exert its collagenase‐inducing effects extra‐cellularly, perhaps through transmembrane matrix receptors, our observation that this neurotoxin dramatically alters protein synthesis in synovial fibroblasts suggests that direct effects on cell metabolism may also play a role in acute acryl‐amide intoxication.
The cytoskeletal system of mammalian primitive erythrocytes: Studies in developing marsupials
Authors: Cohen William D. , Cohen Marion F. , Hugh Tyndale‐Biscoe C. , Vandeberg John L. , Ralston Gregory B.
Published: February 2005

Seeking to resolve conflicting literature on cytoskeletal structure in mammalian “primitive” generation erythrocytes, we have utilized the circulating blood of developing marsupials. In young of the Tammar Wallaby (Macropus eugenii) and the Gray Short‐tailed Opossum (Monodelphis domestica), relatively large, nucleated primitive erythrocytes constituted nearly 100% of the circulating population of birth (= day 0) and in fetuses (Tammar) several days before birth. These cells were discoidal or elliptical, and flattened except for a nuclear bulge. Their cytoskeletal system, consisting of a marginal band of microtubules enclosed within a cell surface‐associated network (membrane skeleton), closely resembled that of non‐mammalian vertebrate erythocytes. By day 2 or 3, much smaller anucleate erythrocytes of “definitive” morphology, lacking marginal bands, appeared in abundance. These accounted for >90% of the circulating population of both species by day 6–8. Non‐nucleated erythrocytes of a different type, constituting 1–6% of the cells in most blood samples up to day 7, were identified as anucleate primitives on the basis of size, shape, and presence of a marginal band. Thus, loss of erythrocyte nuclei in mammals appears to begin earlier than generally recognized, i.e., in the primitive generation. Counts of these anucleate primitives in young of various ages implicated nucleated primitives as their probable source. Pointed erythrocytes, occasionally found in younger neonates of both species, occurred in greatest number in fetuses (Tammar) prior to birth. This is in accord with previous work on non‐mammalian vertebrates suggesting that such cells are morphogenetic intermediates. The results confirm the long‐suspected similarity between mammalian primitive erythrocytes and the nucleated erythrocytes of all non‐mammalian vertebrates.
On metachronism in ciliary systems: A model describing the dependence of the metachronal wave properties on the intrinsic ciliary parameters
Authors: Gheber Larisa , Priel Zvi
Published: February 2005

A mathematical model is proposed to explain the dependence of the direction and the length of the metachronal wave on parameters that characterize the ciliary beat, the dimensions of the cilia, and the geometry of their arrangement on the ciliated surface. The metach/onal wave is decomposed into two mutually perpendicular components, which are chosen in such a way that the direction of one of them is in the direction of the effective stroke. The magnitudes of the two components are determined by using the concept of the time of delay between adjacent cilia. The properties of the metachronal wave are then calculated as a function of the ciliary parameters.

The results obtained with the present model predict that the direction of the wave propagation is strongly dependent on the type of metachronism in the direction of the effective stroke and the polarization in time and in space of the ciliary beat. The metachronal wavelength is found to depend on four parameters: the ciliary length, the angle of the arc projected on the cell surface by the ciliary tip during the recovery stroke, the degree of asymmetry of ciliary beat, and the portion of the cycle occupied by the pause. The metachronal wavelength is also found to be only weakly dependent on the ciliary frequency.

At this stage there exists relatively little experimental information with which t o characterize fully the metachronal properties of ciliary systems. Even when only partial information exists, the model allows prediction, to within a certain range, of the direction of the wave propagation. It also suggests a possible mechanism for the influence of changes in environmental conditions on wave direction and wavelength. In severalcases in which full information does exist, good agreement between the experimental findings and the predictions of the model is found. According to this model it will be worthwhile to invest more effort in measuring the time and space polarization of ciliary beating and the times of delay between cilia.
Quantitative relationships between single‐cell and cell‐population model parameters for chemosensory migration responses of alveolar macrophages to C5a
Authors: Farrell Brian E. , Daniele Ronald P. , Lauffenburger Douglas A.
Published: February 2005

Phenomenological parameters from a mathematical model of cell motility [1] are used to quantitatively characterize chemosensory migration responses of rat alveolar macrophages migrating to C5a in the linear under‐agarose assay, simultaneously at the levels of both single cells and cell populations. This model provides theoretical relationships between single‐cell and cell‐population motility parameters. Our experiments offer a critical test of these theoretical linking relationships, by comparison of results obtained at the cell population level to results obtained at the single‐cell level.

Random motility of a cell population is characterized by the random motility coefficient, μ (analogous to a particle diffusion coefficient), whereas single‐cell random motility is described by cell speed, s, and persistence time, P (related to the period of time that a cell moves in one direction before changing direction). Population chemotaxis is quantified by the chemotactic sensitivity, χo, which provides a measure of the minimum attractant gradient necessary to elicit a specified chemotactic response. Single‐cell chemotaxis is characterized by the chemotactic index, CI, which ranges from 0 for purely random motility to 1 for perfectly directed motility. Measurements of cell number versus migration distance were analyzed in conjunction with the phenomenological model to determine the population parameters while paths of individual cells in the same experiment were analyzed in order to determine the single‐cell parameters.

The parameter μ shows a biphasic dependence on C5a concentration with a maximum of 1.9 × 10−8 cm2/sec at 10−11 M C5a and relative minima of 0.86 × 10−8 cm2/sec at 10−7 M C5a and 1.1 × 10−8 cm2/sec in the absence of C5a; s and P remain fairly constant with C5a concentration, with s ranging from 2.1 to 2.5 μm/min and P varying from 22 to 32 min. χo is equal to 1.0 × 10−6 cm/receptor for all C5a concentrations tested, corresponding to 60% correct orientation for a difference of 500 bound C5a receptors across a 20 μm cell length. The maximum CI measured was 0.2.

Values for the population parameters μ and χo were calculated from single‐cell parameter values using the aforementioned theoretical linking relationships. The values of μ and χo calculated from single‐cell parameters agreed with values of μ and χo determined independently from population migrations, over the full range of C5a concentrations, confirming the validity of the linking equations. Experimental confirmation of such relationships between single‐cell and cell‐population parameters has not previously been reported.
Immunocytochemical identification of cytoskeletal linkages to smooth muscle cell nuclei and mitochondria
Authors: Stromer Marvin H. , Bendayan Moise
Published: February 2005

In avian smooth muscle cells, desmin‐containing intermediate filaments (IFs) are a prominent component of the cytoskeleton and are readily seen in several domains, inclu‐ding the axial intermediate filament bundle (IFB). Both the nucleus and some of the mitochondria are partly surrounded by elements of the IFB. By using anti‐desmin and protein‐A‐colloidal gold labeling, we have identified intermediate filaments that form linkages with the nuclear envelope and with mitochondria. These linkage regions seem to occupy a proportionately greater part of the mitochondrial surface than of the nuclear envelope. The existence of these linkages in smooth muscle cells is consistent with results that support similar linkages to mitochondria and other cellular structures in various cells that contain either vimentin or keratin IFs. These linkages could functionally restrain or assist in homeostatically restoring organelles to their normal position after the rearrangement that accompanies the substantial shortening of smooth muscle cells.
Distribution of microtubules containing post‐translationally modified α‐tubulin during drosophila embryogenesis
Authors: Warn R. M. , Harrison A. , Planques V. , Robert‐Nicoud N. , Wehland J.
Published: February 2005

The distribution of microtubules (MTs) enriched in detyrosinated α‐tubulin (Glu‐tubulin) was studied in Drosophila embryos by immunofluorescence micro‐scopy by using a monoclonal antibody (ID5) which was raised against a 14‐residue synthetic peptide spanning the carboxyterminal sequence of Glu‐tubulin (Wehland and Weber: J. Cell Sci. 88:185‐203, 1987). While all MT arrays contained tyrosinated α‐tubulin (Tyr‐tubulin), MTs rich in Glu‐tubulin were not found during early stages of development even by using an image intensification camera. Elevated levels of microtubular Glu‐tubulin were first detected after CNS condensation in neurone processes. In addition, sperm tails, which remained remarkably stable inside the embryo until late stages of development, were decorated by ID5. This was in marked contrast to the distribution of microtubule arrays containing acetylated α‐tubulin, which could already be detected during the cellular blastoderm stage. Additional experiments with taxol suggested that the absence of MTs rich in Glu‐tubulin during early stages of development was not due to the rapid turnover rate of MTs, which would be too fast for α‐tubulin to be detyrosinated. The possible significance of the differential detyrosination and acetylation of microtubules during development is discussed.
Identification and localization of proteins immunologically related to intermediate filament proteins in sea urchin eggs and embryos
Authors: St‐Pierre Johanne , Dufresne Louise
Published: February 2005

Sea urchin spermatozoa, eggs, and embryos were labeled with the universal antibody against the intermediate filament proteins (anti‐IFA) described by Pruss et al. [Cell 27:419–428, 1981] and with anti‐beta‐tubulin. Localization of these antibodies was by indirect immunofluorescence microscopy. Cytoskeleton of unfertilized eggs, prepared according to a procedure adapted from Kane [Exp. Cell Res. 162:495–506, 1986] or as described by Dufresne et al. [Biochem. Cell Biol. 66:780–791, 1988], and reacted with the anti‐IFA demonstrate a uniformly stained background except for the nuclear areas, which appear as dark rings. During the first cell cycle, the anti‐IFA staining pattern coincides with that of spindle‐associated tubulin but not with the cortical pattern of microtubules. Swimming embryos reacted with the anti‐IFA show a labeling located on the cilia and within the cytoplasm of each individual cell of the larva. In spermatozoa, the labeling occurs all along the flagellae. Immunoblots of proteins from eggs and embryos reveal one major protein of 117 kDa and sometimes a component of 66 kDa, both of which cosediment with tubulin during the isolation procedure of microtubules described by Vallee and Bloom [Proc. Natl. Acad. Sci. USA 80:6259–6263, 1983]. These data show that proteins homologous to the intermediate filament proteins reported in vertebrate cells are present in both gametes of sea urchins. The specific localization ofthese proteins in the spindle, the flagella, and the cilia suggest that they may play a significant role in the organization and function of the microtubular lattice of the spermatozoa and of the embryo.
Identification of an amino acid substitution in the benA, β‐tubulin gene of Aspergillus nidulans that confers thiabendazole resistance and benomyl supersensitivity
Authors: Jung M. Katherine , Oakley Berl R.
Published: February 2005

We are using molecular genetic techniques to identify sites of interaction β‐tubulin with benzimidizole anti‐microtubule agents. We have developed a marker‐rescue technique for cloning mutant alleles of the benA, β‐tubulin gene of Aspergillus nidulans and have used the technique to clone two mutant benA alleles, benA16 and benA19. These are the only A. nidulans alleles known to confer resistance to the benzimidazole antimicrotubule agent thiabendazole and supersensitivity to other benzimidazole antimicrotubule agents including benomyl and its active breakdown product, carbendazim. benA16 has been shown, moreover, to reduce thiabendazole binding to β‐tubulin. We have sequenced the two mutant alleles and have found that they carry different nucleotide changes that cause the same single amino acid substitution, valine for alanine at amino acid 165. Since thiabendazole and carbendazim differ at only one side chain, the R2 group, we conclude that the region around amino acid 165 is involved in the binding of the R2 group of benzimidazole antimicrotubule agents to β‐tubulin.
Microtubule‐associated proteins from antarctic fishes
Authors: Detrich H. William , Neighbors Bonnie W. , Sloboda Roger D. , Williams Robley C.
Published: February 2005

Microtubules and presumptive microtubule‐associated proteins (MAPs) were isolated from the brain tissues of four Antarctic fishes (Notothenia gibberifrons, N. coriiceps neglecta, Chaenocephalus aceratus, and a Chionodraco sp.) by means of a taxol‐dependent, microtubule‐affinity procedure (cf. Vallee: Journal of Cell Biology 92:435–442, 1982). MAPs from these fishes were similar to each other in electrophoretic pattern. Prominent in each preparation were proteins in the molecular weight ranges 410,000–430,000, 220,000–280,000, 140,000–155,000, 85,000–95,000, 40,000–45,000, and 32,000–34,000. The surfaces of MAP‐rich microtubules were decorated by numerous filamentous projections. Exposure to elevated ionic strength released the MAPs from the microtubules and also removed the filamentous projections. Addition of fish MAPs to subcritical concentrations of fish tubulins at 0–5°C, induced the assembly of microtubules. Both the rate and the extent of this assembly increased with increasing concentrations of the MAPs. Sedimentation revealed that approximately six proteins, with apparent molecular weights between 60,000 and 300,000, became incorporated into the microtubule polymer. Bovine MAPs promoted microtubule formation by fish tubulin at 2–5°C, and proteins corresponding to MAPs 1 and 2 co‐sedimented with the polymer. MAPs from C. aceratus also enhanced the polymerization of bovine tubulin at 33°C, but the microtubules depolymerized at 0°C, We conclude that MAPs are part of the microtubules of Antarctic fishes, that these proteins promote microtubule assembly in much the same way as mammalian MAPs, and that they do not possess special capacities to promote microtubule assembly at low temperatures or to prevent cold‐induced microtubule depolymerization.
The iris diaphragm model of centriole and basal body formation
Authors: Albrecht‐Buehler Guenter
Published: February 2005

This paper suggests that the formation and structure of the microtubular skeleton of centrioles and basal bodies can be derived from the following simple geometric principle. A closed ring of nine microtubular initiation sites defines (1) a template for the packing of 18 additional microtubular initiation sites, and (2) the shape of nine rigid arms. Upon swivelling of each arm around a point located four initiation sites away on the initial ring, the array unfolds in a manner similar to the opening of an iris diaphragm.

As a consequence, the curved shape of the microtubular triplet blades arises together with the clockwise rotational sense of the slanted blades of the centriole or basal body. The final diameter of the centriole (basal body) self‐adjusts. Furthermore, the pitch of the triplet blades, the taper of centrioles and basal bodies, and the change of slant of the blades towards the distal end can be derived. In addition, the model points to a method of replication of pro‐centrioles (pro‐basal bodies). The hypothesis was tested by the fitting of electron microscopical cross sections of centrioles of 3T3 cells to the geometric shapes predicted by the model.
p34cdc2 Kinase is localized to distinct domains within the mitotic apparatus
Authors: Rattner J. B. , Lew John , Wang Jerry H.
Published: February 2005

Antibodies to both the C‐terminal and the N‐terminal regions of the 34 kd serinethreonine specific protein kinase, p34cdc2, were used to study the distribution of this protein in dividing cells and isolated chromosomes of the Indian muntjac. p34cdc2 was found to be present throughout the cytoplasm of dividing cells. In addition, a portion of cellular p34cdc2 was localized to the centrosome, kinetochore, and intercellular bridge and along kinetochore‐to‐pole microtubules during cell division. Tubulin‐denuded metaphase kinetochores retained their association with p34cdc2. The detection of p34cdc2 within a variety of domains of the mitotic apparatus, in addition to the previous reported association with the centrosome [Bailly et al., EMBO J. 8:3985–3995, 1989; Raibowol et al., Cell 57:393–401, 1989] suggests that p34cdc2 may play a role in events associated with anaphases A and B as well as with the transition between interphase and mitosis.
Polypeptides from the myxomycete Physarum polycephalum interacting in vitro with microtubules
Authors: Albertini Catherine , Akhavan‐Niaki Haleh , Wright Michel
Published: February 2005

Microtubule‐interacting proteins have been studied in the lower eukaryote Physarum polycephalum. We show for the first time 1) the presence in Physarum amoebal crude extracts of at least six polypeptides that bind specifically to amoebal microtubules, 2) the binding between these proteins and mammalian microtubules, 3) the heat stability of two of these polypeptides (125 and 235 kDa), 4) the functional properties of a fraction containing a heat‐soluble 125 kDa polypeptide, and 5) the phosphorylation of the 125 kDa polypeptide during two distinct periods of the cell cycle in Physarum synchronous plasmodia, first at late S/early G2 phase and second at late G2/prophase.
Yeast myosin heavy chain mutant: Maintenance of the cell type specific budding pattern and the normal deposition of chitin and cell wall components requires an intact myosin heavy chain gene
Authors: Rodriguez José R. , Paterson Bruce M.
Published: February 2005

Recent studies with myosin heavy chain mutants in the slime mold Dictyostelium discoideum and the yeast Saccharoymyces cerevisiae indicate that the myosin heavy chain gene is not essential for cell survival under laboratory growth conditions. However, cells lacking a normal myosin heavy chain gene demonstrate substantial alterations in growth and cell division. In this study, we report that a disruption mutant in the rod portion of the yeast myosin heavy chain gene, MYOl, produces abnormal chitin distribution and cell wall organization at the mother‐bud neck in a high proportion of dividing cells. It is suggested that this phenotype is the cause of the cell division defect and the osmotic sensitivity of yeast MYOl mutants. In the absence of a normal MYOl polypeptide, yeast cells alter their cell type specific budding pattern. It is concluded that an intact myosin heavy chain gene is required to maintain the cell type specific budding pattern and the correct localization and deposition of chitin and cell wall components during cell growth and division.
Calyculin‐A increases the level of protein phosphorylation and changes the shape of 3T3 fibroblasts
Authors: Chartier Lynn , Rankin Lucinda L. , Allen Ronald E. , Kato Yuko , Fusetani Nobuhiro , Karaki Hideaki , Watabe Shugo , Hartshorne David J.
Published: February 2005

Calyculin‐A, an inhibitor of type 1 and 2A phosphatases, was applied extracel‐lularly to 3T3 fibroblasts. At 0.1 μM, calyculin‐A caused a marked increase in protein phosphorylation in both the cytosolic and insoluble cellular fractions. This effect was independent of external Ca2+. An immunoprecipitate, formed with an antibody to myosin, contained several cytoskeletal components. Increased phos‐phorylation following treatment with calyculin‐A was observed in vimentin, the 20‐kD myosin light chain, and an unidentified 440‐kD component. An enhanced level of vimentin phosphorylation was found in intermediate filament preparations from treated cells.

Calyculin‐A also caused marked shape changes of 3T3 cells. Within minutes after addition of calyculin‐A (0.1 μM) cells became rounded and lost attachment to the substratum. Stress fibers, intermediate filaments, and microtubules, prominent in the attached control cells, were not evident in the rounded cells. Shape changes were reversible and after removal of calyculin‐A the rounded cells attached to the substratum, resumed a flattened shape, and were active mitotically. In the cells treated with calyculin‐A an unusual “ball‐like” structure was observed with transmission electron microscopy. This unique structure was 2–3 μM in diameter and was located close to the nucleus.

The use of calyculin‐A adds further support to the idea that cell shape is controlled, at least in part, by concerted actions of a kinase‐phosphatase couple.
Dilution‐induced disassembly of microtubules: Relation to dynamic instability and the GTP cap
Authors: Voter William A. , O'Brien E. Timothy , Erickson Harold P.
Published: February 2005

Microtubules were assembled from purified tubulin in the buffer originally used to study dynamic instability (100 mM PIPES, 2 mM EGTA, 1 mM magnesium, 0.2 mM GTP) and then diluted in the same buffer to study the rate of disassembly. Following a 15‐fold dilution, microtubule polymer decreased linearly to about 20% of the starting value in 15 sec. We determined the length distribution of microtubules before dilution, and prepared computer simulations of polymer loss for different assumed rates of disassembly. Our experimental data were consistent with a disassembly rate per microtubules of 60 μm/min. This is the total rate of depolymerization for microtubules in the rapid shortening phase, as determined by light microscopy of individual microtubules (Walker et al.: Journal of Cell Biology 107:1437–1448, 1988). We conclude, therefore, that microtubules began rapid shortening at both ends upon dilution. Moreover, since we could detect no lag between dilution and the onset of rapid disassembly, the transition from elongation to rapid shortening apparently occurred within 1 sec following dilution. Assuming that this transition (catastrophe) involves the loss of the GTP cap, and that cap loss is achieved by the sequential dissociation of GTP‐tubulin subunits following dilution, we can estimate the maximum size of the cap based on the kinetic data and model interpretation of Walker et al. The cap is probably shorter than 40 and 20 subunits at the plus and minus ends, respectively.
Microtubules in the fission yeast Schizosaccharomyces pombe contain only the tyrosinated form of α‐tubulin
Authors: Alfa Caroline E. , Hyams Jeremy S.
Published: February 2005

The state of tubulin tyrosination in the fission yeast Schizosaccharomyces pombe was investigated using a combination of indirect immunofluorescence microscopy and Western blotting. Antibodies specific for the tyrosinated form of α‐tubulin stained all microtubule arrays in wild type cells and recognised the two α‐tubulin polypeptides in Western blots of cell extracts enriched for tubulin by DEAE‐Sephadex chromatography. Antisera that specifically recognised the detyrosinated, glu, form, on the other hand, gave consistently negative results, both in cells undergoing rapid exponential growth and in those allowed to accumulate in stationary phase. Neither the “ageing” of microtubules, by arresting cells at different points (late G1 or G2/M) in the cell division cycle, nor stabilising them, using D2O, lead to any detectable tubulin detyrosination. These results suggest that S. pombe lacks the carboxypeptidase that carries out the tubulin detyrosination reaction. This is the first report of an organism that possesses the correct C‐terminal α‐tubulin sequence yet fails to carry out this post‐translational modification. The implication of this novel finding for the biological role of these events is discussed.