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M Schliwa

Publications and source records attributed to M Schliwa.

At least 55 records · Page 3Linked to original sources
PubMed1997

Sequence analysis and immunofluorescence study of alpha- and beta-tubulins in Reticulomyxa filosa: implications of the high degree of beta2-tubulin divergence.

We have cloned and sequenced 2 alpha- and 2 beta-tubulin isoforms from the giant freshwater amoeba Reticulomyxa filosa. The microtubules of this organism exhibit some unusual properties, including the highest rates of assembly and disassembly known and the inability to be stabilized by taxol. The cloned alpha-tubulins show a high degree of identity when compared to an alpha-tubulin consensus sequence. The beta-tubulins, however, are more divergent, the beta2-tubulin being the most unusual beta-tubulin found so far. The deduced amino acid sequence of beta2 shows 55% identity to a beta-tubulin consensus sequence. It also features 51 unique exchanges which cluster in the C-terminal half of the molecule. Several unique exchanges and two insertions occur in regions adjacent to, or directly implicated in, conserved beta-tubulin functions. A phylogenetic analysis places the beta-tubulins of R. filosa in the vicinity of beta-tubulins from fungi and slime molds. Monoclonal and polyclonal antibodies raised against R. filosa tubulins show that the electrophoretic mobility of alpha- and beta-tubulins is reversed with respect to tubulins from most other sources. Immunofluorescence experiments reveal a ubiquitous distribution of both beta-tubulins in the amoebal network. Our observations suggest possible links between the aberrant primary structure of the beta2-tubulin and the unusual properties of R. filosa microtubules.

Amino Acid Sequence↗
PubMed1997

Unusual distribution of gamma-tubulin in the giant fresh water amoeba Reticulomyxa filosa.

We have isolated a cDNA encoding gamma-tubulin of Reticulomyxa. The deduced amino acid sequence revealed a high homology to known gamma-tubulin sequences from other organisms, underlining the hypothesis of conserved functions for gamma-tubulin in the cell. After introduction of restriction sites by site-directed mutagenesis and polymerase chain reaction (PCR), we cloned the cDNA into a bacterial expression vector. Recombinant gamma-tubulin was expressed in considerable amounts. Polyclonal antibodies raised against the recombinant material recognized in Western blots the bacterially expressed gamma-tubulin and in the amoeba crude extract a single band representing most likely the amoebal gamma-tubulin. In immunofluorescence studies the antibody showed a diffuse staining in the cytoplasm and no enrichment at centrosome-like structures. Because the assembly of centrosome-independent microtubules can be observed in the far extensions of the amoeba, the data suggest that in this amoeba gamma-tubulin might be involved in the centrosome-independent assembly of microtubules.

Amino Acid Sequence↗
PubMed1996

Characterization of the biophysical and motility properties of kinesin from the fungus Neurospora crassa.

Neurospora kinesin (Nkin) is a distant relative of the family of conventional kinesins, members of which have been identified in various animal species. As in its animal counterparts, Nkin most likely is an organelle motor. Because it is a functional homologue of the kinesin heavy chain of higher eukaryotes, its biophysical and motility properties were compared with those of other conventional kinesins. Purified Nkin behaves as a homodimeric complex composed of two subunits of a 105-kDa polypeptide. Based on its hydrodynamic properties (Stokes radius and sedimentation coefficient), Nkin is an elongated molecule, although it is more compact than its animal counterparts. A detailed comparison of the motility properties of Nkin with those of animal conventional kinesins reveals similarities and some intriguing differences. Nkin is less effective than other kinesins in the use of natural nucleoside triphosphates but responds to a selection of ATP analogues in a similar fashion as mammalian kinesin. Even in the presence of saturating concentrations of ATP, Nkin is significantly more sensitive to ADP or tripolyphosphate than other kinesins. Both the ATP-driven microtubule gliding activity and the microtubule-stimulated ATPase activity of Nkin obey Michaelis-Menten kinetics. Surprisingly, however, the Km values for both these activities are approximately an order of magnitude higher than those of other kinesins. Whether the low affinity for ATP suggested by these high Km values is related to the high rate of motility remains to be determined.

Adenosine Triphosphatases↗
PubMed1996

Viscoelastic properties of F-actin solutions in the presence of normal and mutated actin-binding proteins.

A minimal level of viscoelasticity in the cytoskeleton is an essential prerequisite of cellular motility. To determine the influence of the F-actin crosslinking proteins alpha-actinin and actin-binding protein (ABP)120 gelation factor from Dictyostelium discoideum on the properties of actin gels we used a torsion pendulum to measure directly viscoelastic changes of the filamentous networks. Using the capping proteins severin and DS151 to control actin filament length, both crosslinkers were found to increase the elasticity and the viscosity of F-actin solutions. In the case of alpha-actinin, this activity was completely blocked by micromolar concentrations of Ca2+. The inhibitory functions of the two EF hands of alpha-actinin were further investigated by introducing point mutations into either one or both of the Ca(2+)-binding regions. Mutations in the Ca(2+)-coordinating amino acid residues in the first or in both EF hands left the dynamic storage and loss moduli of the F-actin solution unaltered, independent of the Ca2+ concentration. However, alpha-actinin mutated in the second EF hand increased the viscoelasticity of actin gels like the wild-type protein in the absence of Fa2+. The ABP120 gelation factor exhibited only negligible differences to alpha-actinin in viscometry measurements, whereas its impact on the ratio G"/G' (the ratio of energy lost compared to elastically stored during a deformation) of F-actin solutions was clearly smaller than that of alpha-actinin. We conclude from these data that: (i) a torsion pendulum is an excellent tool to determine small changes of activity in normal and mutated actin-binding proteins, (ii) the first EF hand of alpha-actinin is crucial for its crosslinking function, and (iii) the viscoelastic properties of F-actin gels crosslinked by either alpha-actinin or the ABP120 gelation factor are different.

Actinin↗
PubMed1996

Mechanical perturbation elicits a phenotypic difference between Dictyostelium wild-type cells and cytoskeletal mutants.

To determine the specific contribution of cytoskeletal proteins to cellular viscoelasticity we performed rheological experiments with Dictyostelium discoideum wild-type cells (AX2) and mutant cells altered by homologous recombination to lack alpha-actinin (AHR), the ABP120 gelation factor (GHR), or both of these F-actin cross-linking proteins (AGHR). Oscillatory and steady flow measurements of Dictyostelium wild-type cells in a torsion pendulum showed that there is a large elastic component to the viscoelasticity of the cell pellet. Quantitative rheological measurements were performed with an electronic plate-and-cone rheometer, which allowed determination of G', the storage shear modulus, and G", the viscous loss modulus, as a function of time, frequency, and strain, respectively. Whole cell viscoelasticity depends strongly on all three parameters, and comparison of wild-type and mutant strains under identical conditions generally produced significant differences. Especially stress relaxation experiments consistently revealed a clear difference between cells that lacked alpha-actinin as compared with wild-type cells or transformants without ABP120 gelation factor, indicating that alpha-actinin plays an important role in cell elasticity. Direct observation of cells undergoing shear deformation was done by incorporating a small number of AX2 cells expressing the green fluorescent protein of Aequorea victoria and visualizing the strained cell pellet by fluorescence and phase contrast microscopy. These observations confirmed that the shear strain imposed by the rheometer does not injure the cells and that the viscoelastic response of the cell pellet is due to deformation of individual cells.

Actinin↗
PubMed1996

A novel structural component of the Dictyostelium centrosome.

The microtubule-organizing center of D. discoideum is a nucleus-associated body (NAB) that consists of a multilayered, box-shaped core embedded in an amorphous corona from which the microtubules emerge. The composition of the NAB is still largely unresolved. Here we have examined a high molecular mass component of the NAB which was identified by a monoclonal antibody raised against isolated nucleus/NAB complexes. This antibody recognized a 350 kDa component which is immunologically related to the D. discoideum heavy chain of myosin II. The 350 kDa antigen was localized only at the NAB in interphase cells, while in mitotic cells it may also be found in the vicinity of the NAB as well as in association with the mitotic spindle. Immunogold labeling experiments showed that the protein is part of the NAB corona. This association was not destroyed by treatment with 2 M urea or 0.6 M KCl. The 350 kDa antigen was part of the thiabendazole-induced cytoplasmic microtubule-organizing centers. A direct role in the polymerization of tubulin could not be determined in an in vitro microtubule nucleation assay, whereas antibody electroporation of live cells appeared to interfere with the generation of a normal microtubule system in a subset of cells. Our observations suggest that the 350 kDa antigen is a structural component of the NAB corona which could be involved in its stabilization.

Animals↗
PubMed1996

Flexural rigidity of microtubules measured with the use of optical tweezers.

The flexural rigidity of single microtubules is measured using optical tweezers. Two new methods are presented. In both the optical forces of the laser trap are used to directly manipulate microtubules grown off the ends of Chlamydomonas axonemes. The shapes of the microtubules are observed by video microscopy as the hydrodynamic forces of viscous flow counteract the elastic restoring forces when the microtubules are moved actively relative to the surrounding buffer medium. To determine the flexural rigidity, the bending of a microtubule is analyzed under a given velocity distribution along its length. Microtubules incubated with taxol after polymerization are measured to be more flexible than those without taxol added. On the other hand, MAPs are shown to increase microtubule stiffness.

Animals↗
PubMed1995

The Neurospora organelle motor: a distant relative of conventional kinesin with unconventional properties.

The "conventional" kinesins comprise a conserved family of molecular motors for organelle transport that have been identified in various animal species. Organelle motors from other phyla have not yet been analyzed at the molecular level. Here we report the identification, biochemical and immunological characterization, and molecular cloning of a cytoplasmic motor in a "lower" eukaryote, the Ascomycete fungus Neurospora crassa. This motor, termed Nkin (for Neurospora kinesin), exhibits several unique structural and functional features, including a high rate of microtubule transport, a lack of copurifying light chains, a second P-loop motif, and an overall sequence organization reminiscent of a kinesin-like protein. However, a greater than average sequence homology in the motor domain and the presence of a highly conserved region in the C-terminus identify Nkin as a distant relative of the family of conventional kinesins. A molecular phylogenetic analysis suggests Nkin to have diverged early in the evolution of this family of motors. The discovery of Nkin may help identify domains important for specific biological functions in conventional kinesins.

Adenosine Triphosphatases↗
PubMed1995

Truncation mutagenesis of the non-alpha-helical carboxyterminal tail domain of vimentin reveals contributions to cellular localization but not to filament assembly.

We have investigated the effect of stepwise truncating the carboxyterminal domain ("tail") of the intermediate filament (IF) protein vimentin of the clawed toad, Xenopus laevis, on filament assembly in vitro and, using cell transfection, in vivo and also on the cellular topology of the structures formed. All truncations examined, except the minimal one missing the last 11 amino acids which made the protein more sensitive to changes of ionic strength, did not significantly alter IF assembly in vitro, as judged by electron microscopy, viscometry and determination of viscoelastic properties with a laser-operated torsion pendulum. Stable transfections of vimentin-free mammalian cells with cDNAs encoding these mutations resulted at 28 degrees C, i.e. the permissive temperature for assembly of Xenopus vimentin, in the formation of extended IF bundle arrays. At 37 degrees C, however, the mutants lacking more than the last 35 amino acids could leave the cytoplasm and accumulated in the nucleus, indicating a certain topogenic element is located in the tail and directs cytoplasmic restriction in the wild-type protein although this does not form IFs under these conditions. Transfer to the nucleus is, however, abolished if the IF-consensus motif at the end of the rod domain is removed, suggesting that this part of the molecule also contributes to nuclear location. Similar results were obtained with human vimentin: While the rod entered the nucleus, headless vimentin, unable to form IFs, remained restricted to the cytoplasm owing to its tail domain. In contrast, tailless human vimentin and tailless mouse desmin, which are fully assembly-competent in vitro, both formed extensive IF arrays in the cytoplasm but did not accumulate in the nucleus. We conclude that in class III IF proteins stepwise deletions in the tail, while not considerably altering IF assembly in vitro, can change the topogenesis of IF proteins and structures in the living cell.

Amino Acid Sequence↗
PubMed1994

The mechanical properties of actin gels. Elastic modulus and filament motions.

To address large discrepancies reported in the literature, the viscoelastic properties of gels formed by purified actin filaments have been measured by five different techniques and five different instruments using actin preparations purified separately in four different laboratories. These measurements consistently showed that the elastic shear modulus of 2 mg/ml F-actin is on the order of several hundred pascals, and depends very strongly on the length of the filaments and on the history of the sample prior to measurement. Shortening of actin filaments with gelsolin and mechanical perturbations reduce the shear modulus to low values identical to some reported in the literature, indicating that such perturbations account for low shear moduli and poor responsiveness to filament modifying treatments reported previously. The structures of individual actin filaments within gels very similar or identical to those studied rheometrically were also examined by dynamic light scattering and fluorescence microscopy. Dynamic light scattering data were analyzed by a new method to confirm that actin filaments have no stable associations with each other and fluctuate in solution at a rate governed by the filament bending modulus or persistence length, determined to be approximately 10 microns. Fluorescence microscopy confirmed that applying even small shear stresses to F-actin can orient and rupture the filaments, and that in a minimally perturbed viscoelastic gel, long actin filaments are free to diffuse within a limit of constraints formed by their neighbors. These findings confirm that relatively isotropic F-actin networks are sufficiently strong to stabilize cells.

Actins↗
PubMed1993

Video microscopy of organelle inheritance and motility in budding yeast.

By adapting the time-lapse video microscopy techniques that were developed for larger, more complex cells, to living Saccharomyces cerevisiae cells, intracellular organelle movements were observed. Differential interference contrast optics revealed an organelle transport process in cells treated with mating pheromone. Small particles were observed to travel distances of up to 6 microns at rates of 0.11-0.17 (and in one case 0.80) micron/sec. Overall, the frequency of these motile events was quite low compared to what is observed in cell types traditionally studied by video microscopy. The ability to discern clearly the vacuole and nucleus in budding yeast revealed the dynamics of these organelles and the fact that their movements are carefully orchestrated during the cell cycle. Two types of vacuolar dynamics were observed: 1) interconversion between one large organelle and numerous smaller organelles and 2) the formation of projections that extend from the mother cell's vacuole into the bud. When applied to the study of the many available cytoskeletal and cell cycle mutants, the application of video microscopy to the study of organelle movements in living yeast cells will provide a unique opportunity to determine the molecular mechanisms of intracellular motility and to elucidate the temporal controls over these processes.

Biological Transport↗
PubMed1993

Molecular components of the centrosome.

The centrosome organizes microtubules during both interphase and mitosis and therefore governs fundamental processes in the life of a eukaryotic cell. The past few years have seen a substantial increase in the identification of potential components localized at the centrosome. Although we are still far from achieving a coherent picture of the workings of the centrosome, these recent discoveries are promising first steps towards an understanding of centrosomal functions at the molecular level.

Journal Article↗
PubMed1993

Organelle movements in the wild type and wall-less fz;sg;os-1 mutants of Neurospora crassa are mediated by cytoplasmic microtubules.

The cellular basis of organelle transport in filamentous fungi is still unresolved. Here we have studied the intracellular movement of mitochondria and other organelles in the fungus Neurospora crassa. Four different model systems were employed: hyphae, protoplasts, a cell wallless mutant, and experimentally generated small, flattened cell fragments of the mutant cells. Organelle movements were visualized by DIC optics and computer-enhanced video microscopy. In all cell models the transport of organelles was vectorial and saltatory in nature. The mean velocities for mitochondria, particles and nuclei were 1.4, 2.0, and 0.9 microns/s, respectively. Treatment with 10 microM nocodazole for 30 minutes caused a complete disappearance of microtubules and reversibly blocked directed transport of virtually all organelles, whereas cytochalasin D up to 20 microM was without effect. Correlative video and immunofluorescence microscopy of small fragments of wall-less mutant cells revealed a clear match between microtubule distribution and the tracks of moving organelles. We conclude that organelle movement in the filamentous fungus Neurospora crassa is a microtubule-dependent process.

Actins↗
PubMed1992

Mechanism of centrosome positioning during the wound response in BSC-1 cells.

Locomoting cells are characterized by a pronounced external and internal anterior-posterior polarity. One of the events associated with cell polarization at the onset of locomotion is a shift of the centrosome, or MTOC, ahead of the nucleus. This position is believed to be of strategic importance for directional cell movement and cell polarity. We have used BSC-1 cells at the edge of an in vitro wound to clarify the causal relationship between MTOC position and the initiation of cell polarization. We find that pronounced cell polarization (the extension of a lamellipod) can take place in the absence of MTOC repositioning or microtubules. Conversely, MTOCs will reposition even after lamellar extension and cell polarization have occurred. Repositioning requires microtubules that extend to the cell periphery and is independent of selective detyrosination of microtubules extending towards the cell front. Significantly, MTOCs maintain, or at least attempt to maintain, a position at the cell's centroid. This is most clearly demonstrated in wounded monolayers of enucleated cells where the MTOC closely follows the centroid position. We suggest that the primary response to the would is the biased extension of a lamellipod, which can occur in the absence of microtubules and MTOC repositioning. Lamellipod extension leads to a shift of the cell's centroid towards the wound. The MTOC, in an attempt to maintain a position near the cell center, will follow. This will automatically put the MTOC ahead of the nucleus in the vast majority of cells. The nucleus as a reference for MTOC position may not be as meaningful as previously thought.

Animals↗
PubMed1992

Intracellular cyclic AMP not calcium, determines the direction of vesicle movement in melanophores: direct measurement by fluorescence ratio imaging.

Intracellular movement of vesiculated pigment granules in angelfish melanophores is regulated by a signalling pathway that triggers kinesin and dyneinlike microtubule motor proteins. We have tested the relative importance of intracellular Ca2+ ([Ca2+]i) vs cAMP ([cAMP]i) in the control of such motility by adrenergic agonists, using fluorescence ratio imaging and many ways to artificially stimulate or suppress signals in these pathways. Fura-2 imaging reported a [Ca2+]i elevation accompanying pigment aggregation, but this increase was not essential since movement was not induced with the calcium ionophore, ionomycin, nor was movement blocked when the increases were suppressed by withdrawal of extracellular Ca2+ or loading of intracellular BAPTA. The phosphatase inhibitor, okadaic acid, blocked aggregation and induced dispersion at concentrations that suggested that the protein phosphatase PP-1 or PP-2A was continuously turning phosphate over during intracellular motility. cAMP was monitored dynamically in single living cells by microinjecting cAMP-dependent kinase in which the catalytic and regulatory subunits were labeled with fluorescein and rhodamine respectively (Adams et al., 1991. Nature (Lond.). 349:694-697). Ratio imaging of F1CRhR showed that the alpha 2-adrenergic receptor-mediated aggregation was accompanied by a dose-dependent decrease in [cAMP]i. The decrease in [cAMP]i was both necessary and sufficient for aggregation, since cAMP analogs or microinjected free catalytic subunit of A kinase-blocked aggregation or caused dispersal, whereas the cAMP antagonist RpcAMPs or the microinjection of the specific kinase inhibitor PKI5-24 amide induced aggregation. Our conclusion that cAMP, not calcium, controls bidirectional microtubule dependent motility in melanophores might be relevant to other instances of non-muscle cell motility.

Adrenergic alpha-Antagonists↗
PubMed1991

Microsurgical removal of centrosomes blocks cell reproduction and centriole generation in BSC-1 cells.

We have removed the centrosome from cultured BSC-1 cells by microsurgery, leaving enough cytoplasm with the nucleated cell fragment (karyoplast) to ensure survival and growth. In each experiment, we followed the fate of the karyoplast as well as the anucleate cell fragment (cytoplast) containing the original pair of centrioles. Experimental karyoplasts reestablish a juxtanuclear microtubule-organizing center, an astral array of microtubules, and a compact Golgi apparatus. They enter and presumably complete S phase, and they grow beyond the size of an average BSC-1 cell. However, they do not regenerate centrioles in time periods equivalent to more than 10 cell cycles and do not undergo cell division. Control-operated cells with centrosomes left in the karyoplast progress through the cell cycle, duplicate the centrosome, and form clonal cell colonies. We conclude that the removal of centrioles uncouples cell growth from cell reproduction and impedes centriole biogenesis and centrosome duplication.

Animals↗
PubMed1991

The position of the microtubule-organizing center in directionally migrating fibroblasts depends on the nature of the substratum.

Immunofluorescence and confocal microscopy were used to monitor the positioning of microtubule-organizing centers (MTOCs) during directional migration of chicken embryo fibroblasts on planar substrata and within three-dimensional collagen gels. Homologous assay conditions based on the radial emigration of cells from cell aggregates were used in both cases. Whereas approximately 70% of the cells migrating directionally on glass and at least 60% on other planar substrata have their MTOCs anterior to the nucleus, MTOCs are randomly distributed around the nucleus in cells within collagen gels. The anterior location of the MTOC in cells on glass is attained gradually during the first 4 hr of directional migration. Cells on oriented planar substrata, manufactured by photolithographic etching of narrow parallel grooves into the glass surface, also have a random position of the MTOC, although the cells themselves assume a highly polarized cell shape parallel to the grooves. This environment mimics the partial orientation of the collagen fibers produced by the tractive forces of the cells within collagen networks. These findings demonstrate a difference in MTOC positioning between fibroblasts on planar substrata and within a quasinatural environment.

Actins↗