bluuebunny/crossref_metadata_2025 huggingface dataset:
Actin‐binding proteins regulate the work performed by myosin II motors on single actin filaments
Authors: Janson Lee W. , Sellers James R. , Taylor D. Lansing
Published: February 2005

Regulation of actin/myosin II force generation by calcium [Kamm and Stull, Annu. Rev. Physiol. 51:299–313, 1989] and phosphorylation of myosin II light chains [Sellers and Adelstein, “The Enzymes,” Vol. 18, Orlando, FL: Academic Press, 1987, pp. 381–418] is well established. However, additional regulation of actin/myosin II force generation/contraction may result from actin‐binding proteins [Stossel et al., Ann. Rev. Cell Biol. 1:353–402, 1985; Pollard and Cooper, Ann. Rev. Biochem. 55:987–1035, 1986] as they affect the gel state of the actin cytomatrix [reviewed in Taylor and Condeelis, Int. Rev. Cytol., 56:57–143, 1979]. Regulation of the gel state of actin may determine whether an isotonic or isometric contraction results from the interaction between myosin and actin. We have extended the single actin filament motility assay of Kron and Spudich [Proc. Natl. Acad. Sci. U.S.A. 83:6272–6276, 1986] by including filamin or α‐actinin on the substrate with myosin II to examine how actin‐crosslinking proteins regulate the movements of single actin filaments. Increasing amounts of actin‐crosslinking proteins inhibit filament velocity and decrease the number of filaments moving. Reversal of crosslinking yields increased velocities and numbers of moving filaments. These results support the solation‐contraction coupling hypothesis [see Taylor and Fechheimer, Phil. Trans. Soc. London B 299:185–197, 1982] which proposes that increased crosslinking of actin inhibits myosin‐based contraction. This study also illustrates the potentially varied roles of different actin‐crosslinking proteins and offers a novel method to examine actin‐binding protein activity and their regulation of motility at the single molecule level. © 1992 Wiley‐Liss, Inc.
Association of kinesin with characterized membrane‐bounded organelles
Authors: Leopold Philip L. , McDowall Alasdair W. , Pfister K. Kevin , Bloom George S. , Brady Scott T.
Published: February 2005

The family of molecular motors known as kinesin has been implicated in the translocation of membrane‐bounded organelles along microtubules, but relatively little is known about the interaction of kinesin with organelles. In order to understand these interactions, we have examined the association of kinesin with a variety of organelles. Kinesin was detected in purified organelle fractions, including synaptic vesicles, mitochondria, and coated vesicles, using quantitative immunoblots and immunoelectron microscopy. In contrast, isolated Golgi membranes and nuclear fractions did not contain detectable levels of kinesin. These results demonstrate that the organelle binding capacity of kinesin is selective and specific. The ability to purify membrane‐bounded organelles with associated kinesin indicates that at least a portion of the cellular kinesin has a relatively stable association with membrane‐bounded organelles in the cell. In addition, immuno‐electron microscopy of mitochondria revealed a patch‐like pattern in the kinesin distribution, suggesting that the organization of the motor on the organelle membrane may play a role in regulating organelle motility. © 1992 Wiley‐Liss, Inc.
Establishment of division plane and mitosis in monoplastidic guard mother cells of Selaginella
Authors: Cleary Ann L. , Brown Roy C. , Lemmon Betty E.
Published: February 2005

Stomatogenesis, the determinate developmental pathway leading to formation of a pair of specialized guard cells, was studied in monoplastidic cells ofSelaginella. Observations of living cells followed by immunofluorescence microscopy of the same cells made it possible to correlate changes in cytoskeletal organization with developmental events. The guard mother cell divides in a plane perpendicular to previous divisions and this shift in polarity is marked by morphogenetic plastid migration, as well as by extensive reorganization of cytoskeletal arrays. The single plastid divides and daughter plastids move to a position opposite each other (incipient spindle poles). The axis defined by the opposing plastids rotates in the cell before becoming fixed in position with polar plastids adjacent to the lateral anticlinal walls. Plastid polarity predicts spindle orientation and the plane of division. Once division polarity is defined by plastid position, which will remain unchanged throughout mitosis and cytokinesis, cortical microtubules become reorganized from radial to longitudinal (relative to the long axis of the leaf). The initially random cortical F‐actin also becomes aligned longitudinally. A wide preprophase band of microtubules and F‐actin is formed at right angles to the spindle axis. Plastid‐based microtubules establish the preprophase spindle and also connect to the preprophase band. The mitotic spindle remains anchored at the polar plastids. After mitosis, a phragmoplast that forms among microtubules emanating from plastids and nuclei develops in the plane marked previously by the preprophase band. Mitosis is completed in 1 h 15 min ± 3 min (mean ± S.E.). © 1992 Wiley‐Liss, Inc.
Antikinetochore antibodies interfere with prometaphase but not anaphase chromosome movement in living PtK2 cells
Authors: Wise Dwayne A. , Bhattacharjee Lakshmisri
Published: February 2005

Injection of CREST antikinetochore antiserum (AKA) containing antibodies to the kinetochore into living prometaphase PtK2 cells decreased chromosome velocity to near zero. Injection of either phosphate‐buffered saline or CREST antiserum without antikinetochore antibodies (antikinetochore negative: AKN) had no effect on prometaphase oscillations. AKA antiserum injected into anaphase cells at the beginning of chromatid separation had no effect on anaphase chromosome velocity, spindle elongation, or cytokinesis. Visible binding of antikinetochore antibodies in prometaphase cells at room temperature occurred between 5 and 15 minutes after injection. Anaphase cells injected at the beginning of chromatid separation had bound antibody at the end of anaphase. AKA antiserum recognizes in Western blots proteins associated with the primary constriction: CENP‐B, ‐C, and ‐D, as reported by other workers. The control antiserum, AKN, does not recognize these proteins. These results imply that the antigens recognized by CREST antibodies are important for chromosome movement. Whether or not these antigens are themselves motor molecules cannot be addressed by the present data. In addition, the results suggest that these antigens are not involved in an important way in anaphase movement. © 1992 Wiley‐Liss, Inc.
LC20 and kinetics of gizzard myosin subfragment‐1: Digestion with papain vs. S. aureus protease
Authors: Drew Jean S. , White Marianne P. , Moos Carl , Stein Leonard A.
Published: February 2005

Previous reports have shown that papain‐digested gizzard subfragment‐1 (PAP‐S1) has a cleaved regulatory light chain (LC20), and Vmax similar to phosphorylated heavy meromyosin (HMM) Greene et al., Biochemistry 22:530–535, 1983; Sellers et al., J. Biol. Chem. 257:13880‐13883, 1982; Umemoto et al., [J. Biol. Chem. 264:1431–1436, 1989], while S. aureus protease‐digested S‐1 (SAP‐S1) has intact LC20, but Vmax closer to that of unphosphorylated HMM [Ikebe and Hartshorne, 1985]. To determine whether intact LC20 inhibits ATPase activity for subfragment‐ 1 (S1), we compared the kinetic properties and structures of unphosphorylated PAP‐S1 and SAP‐S1. SDS‐PAGE showed that SAP‐S1 had 68 and 24 KDa heavy chain and 20 and 17 KDa light chain components. PAP‐S1 (15 minutes digestion at 20°C) also had 68 and 17 KDa bands, but the single 24 KDa band (24HC) was replaced by a group of 22–24 KDa fragments and LC20 was cleaved to a 16 KDa fragment. At 13 mM ionic strength, both PAP‐S1 and SAP‐S1 had Vmax similar to phosphorylated HMM (1.1–1.5 s−1). SAP‐S1 had the same KATPase as phosphorylated HMM (38 μM actin). but KATPase for PAP‐S1 was 3‐fold stronger (11 μM actin). Subsequent digestion of SAP‐S1 with papain did not significantly change Vmax, but as LC20 and 24HC were cleaved, both KATPase and Kbinding strengthened 3‐ to 5‐fold. Thus, intact LC20 did not inhibit, and cleavage of LC20 did not increase Vmax for S1. Rather, papain cleavage of LC20 and 24HC was associated with strengthened actin binding. © 1992 Wiley‐Liss, Inc.
Organization of cortical microfilaments in dividing root cells
Authors: Liu Bo , Palevitz Barry A.
Published: February 2005

In order to assess the possible role of microfilaments (Mfs) in events preceding plant cell division, actin was localized in root cells of Allium cepa and Tradescantia virginiana by immunofluorescence microscopy. The distribution of Mfs was compared to that of microtubules (Mts) by means of dual localizations employing both antiactin and antitubulin. Cycling interphase cells contain Mfs that extend into all regions of the cytoplasm in random fashion. Prior to the rearrangement of the cortical Mt array into the initial broad preprophase band (PPB), the number of Mfs in the cytoplasm decreases, while a new population appears in the cortex. The cortical Mfs, which usually occupy the entire cell surface, are aligned parallel to the cortical Mts. When the initial PPB appears, these Mfs still cover the cortex or are arranged as a broad band encompassing the PPB. As the PPB narrows, the Mfs are also confined to an increasingly restricted zone usually wider than the PPB.than the PPB. When the PPB reaches its narrowest, densest configuration, aligned Mfs are excluded from the band proper, while others appear in flanking regions of the cortex. From prometaphase through anaphase, cortical Mfs are largely restricted to the ends of the cell overlying the spindle poles; they also tend to become more randomly oriented. Little or no actin is present in the spindle. During telophase, the two zones of aligned cortical Mfs over the ends of the cell gradually disappear and are replaced by new interphase networks. These changes provide additional data on the possible control of PPB organization by actin, and in addition indicate that the cortex may be the origin of the actin that aggregates at the spindle poles during cytochalasin treatment. © 1992 Wiley‐Liss, Inc.
Action of tolytoxin on cell morphology, cytoskeletal organization, and actin polymerization
Authors: Patterson Gregory M. L. , Smith Charles D. , Kimura Lucille H. , Britton Bruce A. , Carmeli Shmuel
Published: February 2005

Tolytoxin, a cytostatic, antifungal macrolide produced by blue‐green algae of the genus Scytonema, is a potent, reversible inhibitor of cytokinesis in cultured mammalian cells. Treatment of KB cells with 2–16 nM tolytoxin results in profound morphological changes, beginning with the formation of zeiotic processes and culminating in nuclear protrusion. In L1210 cells, cytokinesis is inhibited by as little as 2 nM tolytoxin, while karyokinesis proceeds normally, resulting in polynucleation. Tolytoxin specifically disrupts microfilament organization in A10 cells, while having no apparent effect on microtubules or intermediate filaments. Tolytoxin inhibited actin polymerization in vitro and also caused the depolymerization or fragmentation of F‐actin in vitro. Tolytoxin exhibits effects that closely resemble those of cytochalasin B but is effective at concentrations 1/50−1/1,000 that of cytochalasin B. © 1993 Wiley‐Liss, Inc.
Protein kinases are required for embryonic neural crest cell galvanotaxis
Authors: Nuccitelli Richard , Smart Tanya , Ferguson James
Published: February 2005

Embryonic quail neural crest cells migrate towards the negative pole of an imposed dc electric field as small as 7 mV/mm (0.4 mV per average cell length). The involvement of protein kinases in the mechanism utilized by these cells to detect and respond to such imposed fields was tested through the use of several kinase inhibitors. Evidence for the involvement of protein kinase C (PKC) included: (1) inhibition of the directed motility by 1 μM sphingosine that was reversed by the addition of the phorbol ester, PMA; (2) stimulation of a faster response to the imposed field by PMA; and (3) inhibition of the directed translocation by 5 μM H‐7. However, another PKC inhibitor, staurosporin, did not inhibit the directed translocation (1 nM‐1 μM). We also found evidence for the involvement of either cAMP‐ or cGMP‐dependent protein kinase. The galvanotactic response was partially inhibited by the addition of 10 μM H‐9 and the response was enhanced in the presence of the phosphodiesterase inhibitor, IBMX. However, the adenylate cyclase stimulant, forskolin, had no significant influence on the directed motility, although it reduced the average cell velocity. While these experiments suggest that cAMP‐ or cGMP‐dependent protein kinase or PKC may be involved in the galvanotaxis response, two other protein kinases appeared not to be required. The myosin light chain kinase inhibitor, ML‐7, had no effect on the directed motility in an imposed field, so myosin light chain kinase may not be required for galvanotaxis. Similarly, 5 μM W‐7 had no significant effect on the directed translocation, suggesting that calmodulin‐dependent protein kinase is not involved.

Interestingly, the continuous activity of a protein kinase is apparently not required for the directed translocation response. The addition of the PKC and cAMP‐dependent protein kinase inhibitor, H‐7, after the cells had been exposed to the field for 1 hour, had no effect on the subsequent directed translocation. Thus, for these inhibitors to block the directed translocation, they must be present at the same time as the initial field application. This implies that an integral step in the cellular response mechanism for galvanotaxis involves the stimulation of a protein kinase whose effect is long lasting. © 1993 Wiley‐Liss, Inc.
Morphology of nexin links in relation to interdoublet sliding in the sperm flagellum
Authors: Hakan Bozkurt H. , Woolley David M.
Published: February 2005

In this work, we examine whether the “nexin” linkages of the flagellum can extend in length to accommodate interdoublet sliding. Flagellar bends of large angle were induced in bull spermatozoa by hypotonic treatment. It is argued that this produces large interdoublet displacements that are, nevertheless, still within physiological limits. Such flagella were examined by the rapid‐freeze, deep‐etch techique and the nexin linkages identified by their position in relation to the inner dynein arms and by their straplike, bipartite, morphology. They were found to bridge perpendicularly (or occasionally at an angle) between the A‐ and B‐tubules of adjacent doublets. The nexin linkages were no more than ∼20 nm in length, even in regions in which ∼200 nm of sliding could be inferred. Variable registration between adjacent nexin rows gave some further support to the assumption that sliding had indeed taken place. From this, it is concluded that elastic deformation of the links, such as would accommodate interdoublet sliding, does not occur; some form of displacement must occur between nexin and the adjacent B‐tubule. © 1993 Wiley‐Liss, Inc.
Dynamics of microfilaments are similar, but distinct from microtubules during cytokinesis in living, dividing plant cells
Authors: Zhang Dahong , Wadsworth Patricia , Hepler Peter K.
Published: February 2005

The development and dynamics of the phragmoplast cytoskeleton have been analyzed in living stamen hair cells of Tradescantia. Microtubules and actin microfilaments have been identified by microinjecting either carboxyfluorescein labeled brain tubulin or rhodamine phalloidin. Examination with the confocal laser scanning microscope has permitted sequential imaging of the fluorescent cytoskeletal elements in single living cells progressing through division. Phragmoplast microtubules initially emerge through the lateral coalescence of preexisting interzone microtubules. As cytokinesis progresses, these tightly clustered microtubules shorten in length and expand centrifugally toward the cell periphery. By contrast, the phragmoplast microfilaments appear to arise de novo in late anaphase in close association with the proximal surfaces of the reconstituting daughter nuclei. The microfilaments are oriented parallel to the microtubules but conspicuously do not occupy the equatorial region where microtubules interdigitate and where the cell plate vesicles aggregate and fuse. As development proceeds the microfilaments shorten in length and expand in girth, similar to microtubules, although they remain excluded from the cell plate region. In terminal phases of cell plate formation, microtubules degrade first in the central regions of the phragmoplast and later toward the edges, whereas microfilaments break down more uniformly throughout the phragmoplast. © 1993 Wiley‐Liss, Inc.
Control of profilin and actin expression in muscle and nonmuscle cells
Authors: Babcock Gary , Rubenstein Peter A.
Published: February 2005

Profilin is a small G‐actin binding protein implicated in sequestering actin monomers in vivo. We have quantitated profilin and actin expression in human hepatoma HepG‐2 cells and in two mouse myogenic cell lines, BC3H1 and C2C12, to determine whether the expression of profilin and the expression of nonmuscle isoactin or total actin are co‐regulated. During differentiation of both muscle cell types, profilin and nonmuscle actin expression decrease in a coordinate manner as shown by measurements of steady state mRNA and newly synthesized protein. In human hepatoma HepG‐2 cells, the twofold increase in actin synthesis observed after 24 hours of exposure to cytochalasin D did not result in an increase in profilin synthesis. Thus, profilin and actin expression are not coregulated in all cells. To determine if there is sufficient profilin to sequester a large portion of cellular G‐actin, we measured total profilin and G‐actin levels in the three cell types. In each case, profilin accounted for less than 10% of the total G‐actin on a molar basis. Thus, profilin is not responsible for total G‐actin sequestration in these cells. Finally, using poly‐L‐proline affinity chromatography, we showed that, in the cell types tested, less than 20% of the poly‐L‐proline purified profilin existed as a complex with G‐actin. The profilin in these cells may be interacting with cellular components other than actin. © 1993 Wiley‐Liss, Inc.
Interaction of brain mitochondria with microtubules reconstituted from brain tubulin and MAP2 or TAU
Authors: Jung D. , Filliol D. , Miehe M. , Rendon A.
Published: February 2005

To explore the behaviour of microtubule‐associated proteins, MAP2 and TAU in the interactions of mitochondria with microtubules, an homologous acellular system has been reconstituted with organelles isolated from rat brain. We have established a quantitative in vitro binding assay based on the cosedimentation of 125I‐labeled microtubules with mitochondria. We found that binding of microtubules to mitochondria was concentration dependent and saturable. Binding was insensitive to ATP. A comparison of taxol‐stabilized microtubules prepared from MAP‐free tubulin or tubulin coated with TAU or MAP2 showed that the microtubule‐associated proteins diminished, or reduced to background levels, the formation of complexes with mitochondria. In contrast, the amount of MAP‐free taxol microtubules that cosedimented with mitochondria increased two‐ and six‐fold when mitochondria were coated with MAP2 or TAU. These studies suggest that the two major brain MAPs could have a crosslinking or a spacing role, depending on their organelle localization. © 1993 Wiley‐Liss, Inc.
Actin in the merozoite of the malaria parasite, Plasmodium falciparum
Authors: Field S. J. , Pinder Jennifer C. , Clough Barbara , Dluzewski A. R. , Wilson R. J. M. , Gratzer W. B.
Published: February 2005

Merozoites of the human malaria parasite, Plasmodium falciparum, when treated with cytochalasin B, will attach irreversibly to red cells with formation of a vestigial internal (parasitophorous) vacuole, but they are inhibited from moving into the cell. The existence of an actin‐based motile mechanism is implied. Immunoblotting, peptide mapping and the DNase inhibition assay have been used to show that the merozoite contains actin. It makes up an estimated 0.3% of the total parasite protein and is partitioned in the ratio of about 1:2 between the cytosolic and particulate protein fractions. In the former it is unpolymerised and in the latter filamentous. Most of the anti‐actin‐reactive protein in the soluble fraction and about 20% of that in the pellet has an apparent molecular weight of 55,000 and reacts with an anti‐ubiquitin antibody; it is thus evidently ubiquitinyl actin, or arthrin, which has so far been detected only in insect flight muscle. © 1993 Wiley‐Liss, Inc.
Microtubule rearrangement and bending during assembly of large curved microtubule bundles in mouse cochlear epithelial cells
Authors: Tucker John B. , Paton Calum C. , Henderson Craig G. , Mogensen Mette M.
Published: February 2005

Mature inner pillar cells in the mammalian organ of Corti are curved through about 60°, where they arch over adjacent epithelial cells and the apex of an intercellular space called the tunnel of Corti. This report deals with changes in microtubule organization that are associated with cell bending and tunnel formation during morphogenesis of the mouse organ of Corti.

A large bundle of up to 3,000 microtubules assembles in each inner pillar cell. Microtubule rearrangement occurs about 5 days after bundle assembly begins. The lumen of each initially straight hollow tube‐shaped microtubule bundle is occluded as the bundle becomes more compact and elliptical in cross section. This event anticipates the once‐only bending which subsequently occurs between particular levels (abut 9–19 μm) below the top of a bundle as it curves into its final shape about 2 days later. Microtubule rearrangement presumably facilitates bending which is effected in the plane of lest mechanical resistance parallel to the short axis of a bundle's elliptical cross‐sectional profile.

Precocious bending of bundles has been induced about 1.5 days in advance of the natural event. Abnormal positioning of these prematurely curved bundles indicates that bending is effected by a contractile mechanism located within bundles rather than being a response to externally applied forces. The potential importance of such microtubule‐associated contractions for active modulation of the vibratory response in the cochlea during hearing is considered. © 1993 Wiley‐Liss, Inc.
Cellular automaton model of the actin cytoskeleton
Authors: Dufort Paul A. , Lumsden Charles J.
Published: February 2005

We describe a cellular automaton model of the actin cytoskeleton. The model incorporates spatial and temporal behavior at the macomolecular level and is relevant to the viscous nonequilibrium conditions suspected to occur in vivo. The model include cation and nucleotide binding to actin monomers, actin nucleation and polymerization into filaments, coss‐linking with α‐actinin, monomer sequestration with pfilin, filament severing, capping and nucleation with gelsolin, binding of profilin and gelsolin to membrane‐bound phosphatidylinositide biphosphate (PIP2), and regulation of coss‐linking and severing by changing calcium levels. We derive (1) equations for the molecular trnslation and rotation probabilities required for the cellular automaton simulation in terms of molecular size, shape, cytoplasmic viscosity, and temperature; and (2) equations for the binding probabilities of adjacent molecules in terms of experimentally determined reaction rate constants. The model accurately captures the known characteristics of actin polymerization and subsequent ATP hydrolysis under different cation and nucleotide conditions. An examination of gelation and sol‐gel transitions resulting from calcium regulation of α‐actinin and gelsolin predicts an inhomogeneous distribution of bound α‐actinin and F‐actin. The double‐bound α‐actinin (both ends bound to F‐actin) is tightly bunched, while single‐bound α‐actinin is moderately bunched and unbound α‐actinin is homogeneously distributed. The spatial organization of the α‐actinin is quantified using estimates of fractal dimension. The simulation results also suggest that actin/α‐actinin gels may shift from an isotropic to an amorphous phase after shortening of filaments. The gel‐sol transition of the model shows excellent agreement with the present theory of polymer gels. The close correspondence of the model's predictions with previous experimental and theoretical results suggests that the model may be pertinent to better understanding the spatial and temporal properties of complex cytoskeletal processes. © 1993 Wiley‐Liss, Inc.
Rises of intracellular Ca2+ and pH mediate the initiation of sperm motility by hyperosmolality in marine teleosts
Authors: Oda Shoji , Morisawa Masaaki
Published: February 2005

Spermatozoa of marine teleosts, puffers and flounder, wee completely quiescent when they were washed to remove electrolytic components of the seminal plasma and then diluted in nonelectrolyte solutions isotonic to the seminal plasma. Sperm motility was initiated upon dilution in hypertonic nonelectrolyte solutions. These observations suggest that sperm motility is suppressed by seminal osmolality and motility is triggered solely by the increase in external osmolality which occurs at natural spawning in hypertonic seawater. Extracellular Ca2+ had no influence on the osmolality‐dependent initiation of sperm motility. However, sperm motility was initiated even in isotonic solution when Ca2+ was introduced into the sperm cells by Ca2+ ionophore. Intracellular Ca2+ increased at the osmolality‐dependent initiation of sperm motility under Ca2+ ‐free conditions. These results suggest that the release of Ca2+ from intracellular storage in response to the increase in external osmolality has a key role in the initiation of sperm motility. A transient increase in intracellular pH was also observed at the hyperosmolality‐dependent initiation of sperm motility. Furthermore, initiation of sperm motility was induced even in isotonic solutions when intracellular pH increased by the treatment with ammonium salts. These results suggest that an increase in intracellular pH, as well as the rise in intracellular Ca2+, has an important role in the initiation of sperm motility in marine teleosts. © 1993 Wiley‐Liss, Inc.
Sulphonate buffers affect the recovery of microtubule‐associated proteins MAP1 and MAP2: Evidence that MAP1A promotes microtubule assembly
Authors: Pedrotti Barbara , Soffientini Adolfo , Islam Khalid
Published: February 2005

The influence of two commonly used sulphonate buffers, PIPES and MES, on the in vitro assembly of bovine brain microtubule protein was examined. Microtubule assembly was monitored by turbimetry and, after centrifugation, the polymerised protein was analysed by SDS‐PAGE and western blotting. Assembly in MES when compared with PIPES resulted in a higher recovery of microtubule proteins at both pH 6.4 and pH 6.9 and in an altered protein composition. The buffer pH affected the total amount of protein polymerized but did not significantly affect the protein composition. At both pH conditions the recovery of HMW‐MAPs was markedly increased in MES buffer and this increase was mostly due to an increase in the amount of MAP1. © 1993 Wiley‐Liss, Inc.
Perspectives on tubulin isotype function and evolution based on the observation that Tetrahymena thermophila microtubules contain a single α‐ and β‐tubulin
Authors: Gaertig Jacek , Thatcher Thomas H. , McGrath Kathleen E. , Callahan Rosemary C. , Gorovsky Martin A.
Published: February 2005

We have cloned and sequenced the two β‐tubulin genes of the ciliated protozoan Tetrahymena thermophila. The two genes encode identical 443 amino acid peptides which are 99.7% identical to the β‐tubulin proteins of T. pyriformis and 95% identical to human β1 tubulin. T. thermophila contains only one β‐tubulin gene (Callahan et al., 1984: Cell 36:441–445). Thus, all of the extremely diverse microtubule structures in this unicellular organism can be formed from a single α‐ and a single β‐tubulin peptide. We have also carried out a phylogenetic analysis of 84 complete β‐tubulin peptide sequences. This analysis supports two hypotheses regarding β‐tubulin evolution and function: (1) Multifunctional β‐tubulins are under greater evolutionary constraint than β‐tubulins present in specialized cells or in cells with very few microtubule related functions, which can evolve rapidly; and (2) Cells which form axonemes maintain a homogeneous population of tubulins. © 1993 Wiley‐Liss, Inc.
Myosin heavy chain dephosphorylation during cytokinesis in dividing sea urchin embryos
Authors: Larochelle Denis A. , Epel David
Published: February 2005

Myosin heavy chain (MHC) phosphorylation was examined throughout the period of first cleavage in developing sea urchin embryos. MHC was found to be phosphorylated in these cells and, furthermore, the relative state of myosin phosphorylation was found to decrease as cells progressed through cytokinesis. Following the completion of cytokinesis, the relative state of MHC phosphorylation returned to levels observed in precytokinesis cells. The above results were obtained with myosin immunoprecipitated from whole cell lysates. In order to specifically examine the phosphorylation, state of MHC in the cleavage furrow, a protocol was developed for the isolation of intact contractile rings from dividing sea urchin embryos. MHC was immunoprecipitated from isolated contractile rings and the relative phosphorylation state of this MHC was compared with that of MHC isolated from whole cell lysates prepared at the same time. MHC from isolated contractile rings was found to be significantly less phosphorylated than total cellular MHC, suggesting a role for MHC dephosphorylation during cytokinesis. © 1993 Wiley‐Liss, Inc.
Distribution of F‐actin elongation sites in lysed polymorphonuclear leukocytes parallels the distribution of endogenous F‐actin
Authors: Redmond Tim , Zigmond Sally H.
Published: February 2005

We compared, on lysed polymorphonuclear leukocytes (PMNs), the spatial distributions of sites that nucleate actin polymerization with the spatial distribution of endogenous F‐actin. Sites nucleating polymerization of exogenous actin were detected by incubating lysed cells with rhodamine‐labeled G‐actin under polymerizing conditions. Endogenous F‐actin was stabilized and stained by lysis of cells into fluorescein‐labeled (FITC) phalloidin. We found the distributions of rhodamine and fluorescein intensities in a given cell, resting or stimulated with chemoattractant, to be similar. Thus, after lysis the number of sites able to nucleate actin polymerization is proportional to the local F‐actin concentration.

Quantitative fluorescence microscopic analysis also demonstrated that (1) if cells were stimulated with chemoattractant shortly before lysis, the total fluorescence per cell of both fluorophors went up; (2) if peptide was diluted shortly before lysis, the endogenous F‐actin in the lamellae was dramatically reduced, but nucleation sites persisted, giving a high rhodamine to fluorescein ratio; and (3) there was a small increase in the ratio of rhodamine (exogenously grown actin) to fluorescein (endogenous F‐actin) in a region near the lamellar/endoplasm border, centripetal to regions of the highest concentration of endogenous F‐actin.

The rhodamine signal appeared to be due to in situ actin polymerization probably nucleated by existing free barbed ends, since (1) the rhodamine signal increased linearly with time with no detectable lag if the actin concentration was above that of the critical concentration of the barbed end; (2) the rhodamine signal was dramatically reduced if lysates were incubated with gelsolin–actin complex (which stably caps barbed ends), then washed before the rhodamine G‐actin was added; and (3) the number of nucleation sites at the time of lysis is similar to the number of the barbed ends of actin filaments determined by the kinetics of depolymerization [Cano et al., 1991].

The fact that the distribution of exogenous actin polymerization paralleled the endogenous F‐actin suggests that the number of free barbed ends per F‐actin is roughly constant. If all filament ends were free, or if a constant fraction of the filaments ends were free, these data would suggest that the mean filament length is roughly constant throughout the cell. © 1993 Wiley‐Liss, Inc.
Computerized analysis of tumor cells flowing in a parallel plate chamber to determine their adhesion stabilization lag time
Authors: Patton John T. , Mentor David G. , Benson Douglas M. , Nicolson Garth L. , McIntire Larry V.
Published: February 2005

The importance of cell adhesion in a variety of physiological phenomena requires development of an understanding of the factors and molecular mechanisms underlying these behaviors. Cell adhesion is a multistep process involving primary receptor‐ligand interactions followed by secondary events that may lead to the formation of focal contacts. Due to the lack of well‐defined assays to study adhesion stabilization, little is known about this process, except that it may involve signaling events, receptor recruitment, and, as we have demonstrated, covalent peptide cross‐linking by cell membrane‐associated transglutaminase [Menter et al.: Cell Biophys. 18:123–143, 1992]. To study the stabilization process we have developed a dynamic assay employing a parallel plate flow chamber coupled with video microscopy and digital image processing. Our studies utilize wheat germ agglutinin‐selected human metastatic melanoma cell variants that exhibit differences in their experimental metastatic potential and expression of transglutaminase. Using this assay, quantifying cell‐substrate stabilization was found to be quick, reliable, reproducible, and useful in evaluating agents that block this process. © 1993 Wiley‐Liss, Inc.
Rat liver fat‐storing cell lines express sarcomeric myosin heavy chain mRNA and protein
Authors: Ogata Itsuro , Sáez Claudia G. , Greenwel Patricia , de Lourdes Ponce María , Geerts Albert , Leinwand Leslie A. , Rojkind Marcos
Published: February 2005

Fat‐storing cells (FSC, lipocytes, or Ito cells) of liver store vitamin A and are the main producers of extracellular matrix in normal and cirrhotic liver. During liver injury, FSC undergo an activation process characterized by a decrease in vitamin A storage and an increase in cell proliferation and extracellular matrix deposition. This activation process also occurs upon culturing FSC from normal liver. In contrast to most cells of nonmuscle origin, activated FSC express two cytoskeletal proteins normally found in muscle, desmin, and smooth muscle α‐actin. Based on their strategic perisinusoidal location, it has been hypothesized that FSC play a role in regulating blood flow. However, the nature of the contractile elements involved in this process remains to be determined. In this communication we demonstrate the presence of a sarcomeric myosin in proteins solubilized from liver biomatrix. In addition we demonstrate the expression of sarcomeric myosin heavy chain (MHC) mRNA and protein in two FSC clones derived from a CCl4‐cirrhotic rat liver (CFSC). Through cloning the cDNA corresponding to the MHC gene expressed in these cells we demonstrate that it encodes fast IId skeletal MHC and thus represents a marker normally seen in adult muscle. The unexpected expression of an adult stage skeletal muscle molecular motor in FSC from cirrhotic liver is consistent with the proposed specialized contractile capacity of these cells. © 1993 Wiley‐Liss, Inc.
Microtubule sliding in reduced‐amplitude bending waves of Ciona sperm flagella: Resolution of metachronous and synchronous sliding components of stable bending waves
Authors: Brokaw Charles J.
Published: February 2005

Microtubule sliding associated with the bending of reactivated flagella of demembranated spermatozoa of the tunicate, Ciona, has been analyzed using a descriptive model that permits quantitation of metachronous and synchronous components of sliding. Reduced‐amplitude bending waves, obtained by addition of increased salt (K acetate), lithium, or vanadate to the reactivation solutions, have been examined. Increased K acetate can decrease bend angle by as much as 70% with little change in frequency.

In all cases, a decrease in the amplitude, or bend angle, of propagated bends is measured as a decrease in the metachronous component of sliding and is associated with a reduction in the growth of new bends after they begin to propagate during the second half‐cycle of bend development. At higher K acetate concentrations, bend growth during the second half‐cycle of bend development is very strongly reduced and may even become negative. A disparity between the rates of bend growth in the first and second half‐cycles of bend development corresponds to a large amount of synchronous sliding in the distal portion of the flagellum. When the synchronous sliding component is large, the sliding velocity in a propagating bend decreases to near‐0 values and may even reverse its direction as the bend propagates through the mid‐region of the flagellum. Since these large perturbations of sliding velocity do not interfere with regular propagation of bends with nearly constant bend angle, the bend propagation mechanism cannot operate by metachronous control of the velocity of sliding, and is unlikely to operate by local monitoring of either the amount or velocity of sliding. These observations therefore argue against models in which active sliding is regulated by shear or sliding velocity, and make curvature‐controlled models relatively more attractive.

In many cases, a reduction in sliding during bend initiation (the first half‐cycle of development of new bends) also contributes to the decreased amplitude of propagated bends. These changes in bend initiation are similar in both full‐length flagella and in flagella shortened by breakage. The amount of sliding that occurs during bend initiation is relatively independent of the distribution of sliding between metachronous and synchronous components in the distal part of the flagellum. These observations therefore provide additional evidence that bend initiation and bend propagation are independent and separable processes. © 1993 Wiley‐Liss, Inc.
gCap39 is a nuclear and cytoplasmic protein
Authors: Onoda Koji , Yu Fu‐Xin , Yin Helen L.
Published: February 2005

gCap39 is a newly identified member of the Ca2+‐ and polyphosphoinositidemodulated gelsolin family of actin binding proteins which is different from gelsolin in several important respects: it caps filament ends, it does not sever filaments, it binds reversibly to actin, it is phosphorylated in vivo, and it is also present in the nucleus. gCap39 and gelsolin coexist in a variety of cells. To better understand the roles of gCap39 and gelsolin, we have compared their relative amounts and intracellular distributions. We found that gCap39 is very abundant in macrophages (accounting for 0.6% of total macrophage proteins), and is present in 12‐fold molar excess to gelsolin. Both proteins are highly induced during differentiation of the promyelocytic leukemia cell line into macrophages. gCap39 is less abundant in fibroblasts (0.04% total proteins) and is present in equal molar ratio to gelsolin. The two proteins are colocalized in the cytoplasm, but gCap39 is also found in the nucleus while gelsolin is not. Nuclear gCap39 redistributes throughout the cytoplasm during mitosis and is excluded from regions containing chromosomes. Our results demonstrate that gCap39 is a nuclear and cytoplasmic protein which has unique as well as common functions compared with gelsolin. © 1993 Wiley‐Liss, Inc.
Unambiguous classification of microtubule‐ends in vitro: Dynamic properties of the plus‐ and minus‐ends
Authors: Kowalski Richard J. , Williams Robley C.
Published: February 2005

To understand the mechanism of dynamic instability of microtubule growth and shortening, one needs a means of reliably determining the polarity of the microtubules under investigation. Sea urchin sperm‐tail axonemal fragments nucleate the growth of both plus‐ended and minus‐ended microtubules, but their polarity is not apparent by video‐enhanced DIC microscopy. The polarity of a microtubule is usually assessed by observing differences between the rates and lengths of growth and shortening excursions of the two ends. In practice, though, a significant fraction of the population of microtubules displays characteristics intermediate between the average characteristics of either end, thereby escaping classification. Excluding these “intermediate” microtubules from the measured populations introduces bias into the understanding of microtubule dynamic instability. We circumvent this problem by making use of the plus‐end directed movement of the microtubule‐dependent molecular motor kinesin to determine the polarity of any given microtubule unambiguously. Carboxylated‐microspheres coated with kinesin, which are clearly visible by DIC microscopy, were used to determine the polarity of a microtubule. The dynamics were then observed. Kinesin was found to have no marked effect on dynamic instability. By this technique, we show that the distributions of properties that describe microtubule dynamic instability (rates and lengths of growth and shortening as well as frequencies of interconversion between these phases) of plus‐ends overlap to a significant extent with those of minus‐ends. It is this overlap that obscures the usual classification of the ends. Therefore, models describing microtubule dynamic instability need to incorporate the broad and overlapping range of properties of the two ends. © 1993 Wiley‐Liss, Inc.
Three‐dimensional dynamics of pseudopod formation and the regulation of turning during the motility cycle of Dictyostelium
Authors: Wessels Deborah , Vawter‐Hugart Holly , Murray John , Soll David R.
Published: February 2005

Employing a newly developed computer‐assisted system for visualizing and quantitating cell motility in three dimensions, we have examined the 3‐dimensional changes in cell shape and the dynamics of pseuodopod extension during translocation of Dictyostelium amoebae. Amoebae exhibit a 3‐dimensional behavior cycle with an average period of 1.5 min. The cycle includes a transient pseudopod extension phase in the x, y axis followed by a z‐axis expansion phase. Anterior pseudopod extension in the x, y axis is accompanied by a decrease in height, not by uropod retraction. The increase in height is accompanied by uropod retraction. In the pseudopod extension phase in the x, y axes, pseudopods form either anteriorly or laterally, and either on or above the substratum. Pseudopods which initially form on the substratum in almost all cases continue to expand as the anterior end of the cell. In the case of lateral pseuodopods, anteriorization leads to a turn. Approximately half of anterior pseudopod and two‐thirds of lateral pseudopods which initially form above the substratum are retracted. These results suggest that pseudopod‐substratum interaction plays a fundamental role in the regulation of directionality and turning in the translocation phase of the 3‐dimensional behavior cycle. © 1994 Wiley‐Liss, Inc.
Occurrence of fibers and their association with talin in the cleavage furrows of PtK2 cells
Authors: Sanger Jean M. , Dome Jeffrey S. , Hock Rick S. , Mittal Balraj , Sanger Joseph W.
Published: February 2005

PtK2 cells of exceptionally large size were microinjected with fluorescently labeled probes for actin, myosin, filamin, and talin in order to follow the assembly of the contractile proteins into the cleavage furrows. Whereas in cells of normal size, there is usually a diffuse pattern of localization of proteins in the cleavage furrow, in these large, flat cells the labeled proteins localized in fibers in the cleavage furrow. Often, the fibers were striated in a pattern comparable to that measured in the stress fibers of the same cell type. The presence of talin in discrete plaques along fibers in the cleavage furrows of the large cells suggests a further similarity between cleavage furrow and stress fiber structure. The presence of filamin in the cleavage furrows also suggests the possibility of an overlapping mechanism in addition to that of a talin mediated mechanism for the attachment of actin filaments to the cell surfaces in the cleavage furrow. A model is presented that emphasizes the interrelationships between stress fibers, myofibrils, and cleavage furrows. © 1994 Wiley‐Liss, Inc.
Novel 130‐kDa rat liver myosin‐1 will translocate actin filaments
Authors: Williams Roger , Coluccio Lynne M.
Published: February 2005

We have recently purified and characterized from rat liver, polypeptides of 110‐kDa and 130‐kDa which possess several characteristics of myosin‐1 [Coluccio and Conaty: Cell Motil. Cytoskeleton 24:189‐199, 1993]. What roles these myosin‐1 molecules play in hepatocytes is not yet defined. One hypothesis is that they are involved in either intracellular transport or locomotion. As a first step in establishing their function, we have investigated whether these molecules are capable of supporting motility in vitro. Our results clearly demonstrate that the isolated 130‐kDa‐calmodulin complex will translocate filaments at a rate of 0.03‐0.05 μ/sec; motility is inhibited in free calcium ion concentrations above 0.1 μM. This inhibition is reversed with the addition of exogenous calmodulin. These results provide supporting evidence of a motile role for the 130‐kDa‐calmodulin complex in vivo. This is the first demonstration that in higher eukaryotes, myosin‐1 from a tissue other than intestine will support motility. Partial peptide sequence analysis indicates that the 130‐kDa polypeptide resembles the recently described myr 1 [Ruppert et al.: J. Cell Biol. 120:1393‐1403, 1993] or MM1α [Sherr et al.: J. Cell Biol. 1405‐1416, 1993] gene product. © 1994 Wiley‐Liss, Inc.
Role of MAPs and motors in the bundling and shimmering of native microtubules from insect ovarioles
Authors: Hunt Cherryl , Stebbings Howard
Published: February 2005

Bundles of native microtubules isolated from the ovarioles of hemipteran insects are seen to shimmer when observed using dark‐field microscopy. This novel form of microtubule motility becomes even more obvious when the isolated bundles are detergent‐extracted and reactivated. We have studied the nucleotide‐specificity and the drug‐sensitivity of microtubule shimmering in order to obtain information regarding the nature of the motor protein responsible, and to compare its properties with those of previously characterised microtubule motors. The involvement of structural MAPs in the shimmering and in maintenance of microtubule bundles in this system has also been investigated. © 1994 Wiley‐Liss, Inc.