Mechanical analyses of morphological and topological transformation of liposomes.

Liposomes are micro-compartments made of lipid bilayer membranes possessing the characteristics quite similar to those of biological membranes. To form artificial cell-like structures, we made liposomes that contained subunit proteins of cytoskeletons: tubulin or actin. Spherical liposomes were transformed into bipolar or cell-like shapes by mechanical forces generated by the polymerization of encapsulated subunits of microtubules. On the other hand, disk- or dumbbell-shaped liposomes were developed by the polymerization of encapsulated actin. Dynamic processes of morphological transformations of liposomes were visualized by high intensity dark-field light microscopy. Topological changes, such as fusion and division of membrane vesicles, play an essential role in cellular activities. To investigate the mechanism of these processes, we visualized the liposomes undergoing topological transformation in real time. A variety of novel topological transformations were found, including the opening-up of liposomes and the direct expulsion of inner vesicles.

Morphological and topological transformation of membrane vesicles.

Liposomes are micro-compartments made of lipid bilayer membranes withcharacteristics quite similar to those of biological membranes. To formartificial cell-like structures, we generated liposomes that containedsubunit proteins of cytoskeletons: tubulin or actin. Spherical liposomeswere transformed into bipolar or cell-like shapes by mechanical forcesgenerated by the polymerization of encapsulated subunits of microtubules.Disk- or dumbbell-shaped liposomes were developed by the polymerizationof encapsulated actin. Dynamic processes of morphological transformationsof liposomes were visualized by high intensity dark-field lightmicroscopy.Topological changes, such as fusion and division of membrane vesicles,play an essential role in cellular activities. To investigate themechanism of these processes, we visualized in real time the liposomesundergoing topological transformation. A variety of novel topologicaltransformations were found, including the opening-up of liposomes and thedirect expulsion of inner vesicles.

Visualization of the stop of microtubule depolymerization that occurs at the high-density region of microtubule-associated protein 2 (MAP2).

Individual microtubules (MTs) repeat alternating phases of polymerization and depolymerization, a process known as dynamic instability. Microtubule-associated proteins (MAPs) regulate the dynamic instability by increasing the rescue frequency. To explore the influence of MAP2 on in vitro MT dynamics, we correlated the distribution of MAP2 on individual MTs with the dynamic phase changes of the same MTs. MAP2 was modified selectively on its projection region by X-rhodamine iodoacetamide without altering the MT-binding activity. When the labeled MAP2 was added to MTs, the fluorescence was distributed along almost the entire length of individual MTs. However, the inhomogeneity of the distribution gradually became obvious due to the fluorescence bleaching, and the MTs appeared to consist of rapidly bleached portions (RBPs) and slowly bleached portions (SBPs), which were distributed randomly along the MT. By measuring the duration of fluorescence bleaching, the density of MAP2 in SBP was estimated to be approximately 2.5 times higher than the RBP. The average tubulin:MAP2 ratio in SBP was calculated to be 16. When the MT dynamics were observed by dark-field microscopy after determining the MAP2 distribution, rescues were always found to occur only at the SBPs. MTs also displayed intermittent shortening by repeated depolymerization phases separated by pause phases. In these cases, depolymerization phases stopped only at the SBPs. Not every SBP stopped depolymerization, but depolymerization always stopped at an SBP. Taken together, we suggest that there is a minimum density of MAP2 that is necessary to stop depolymerization.

Capabilities of liposomes for topological transformation.

Dynamic behaviors of liposomes caused by interactions between liposomal membranes and surfactant were studied by direct real-time observation by using high-intensity dark-field microscopy. Solubilization of liposomes by surfactants is thought to be a catastrophic event akin to the explosion of soap bubbles in the air; however, the actual process has not been clarified. We studied this process experimentally and found that liposomes exposed to various surfactants exhibited unusual behavior, namely continuous shrinkage accompanied by intermittent quakes, release of encapsulated liposomes, opening up, and inside-out topological inversion.

Ser787 in the proline-rich region of human MAP4 is a critical phosphorylation site that reduces its activity to promote tubulin polymerization.

p34cdc2 kinase-phosphorylation sites in the microtubule (MT)-binding region of MAP4 were determined by peptide sequence of phosphorylated MTB3, a fragment containing the carboxy-terminal half of human MAP4. In addition to two phosphopeptides containing Ser696 and Ser787 which were previously indicated to be in vivo phosphorylation sites, two novel phosphopeptides, containing Thr892 or Thr901 and Thr917 as possible phosphorylation sites, were isolated, though only in in vitro phosphorylation. The role of phosphorylation at Ser696 and Ser787, which were differently phosphorylated during the cell cycle (Ookata et al., (1997). Biochemistry, 36: 15873-15883), was investigated in MT-polymerization, using MAP4 Ser to Glu mutants, which mimic phosphorylation at each site. Mutation of Ser787 to Glu strikingly reduced the MAP4's MT-polymerization activity, while Glu-mutation at Ser696 did not. These results suggest that Ser787 could be the critical phosphorylation site causing MTs to be dynamic at mitosis.

Morphogenesis of liposomes encapsulating actin depends on the type of actin-crosslinking.

To study the morphogenesis of cells caused by the organization of their internal cytoskeletal network, we characterized the transformation of liposomes encapsulating actin and its crosslinking proteins, fascin, alpha-actinin, or filamin, using real-time high-intensity dark-field microscopy. With increasing temperature, the encapsulated G-actin polymerized into actin filaments and formed bundles or gels, depending on the type of actin-crosslinking protein that was co-encapsulated, causing various morphological changes of liposomes. The differences in morphology among transformed liposomes indicate that actin-crosslinking proteins determine liposome shape by organizing their specific actin networks. Morphological analysis reveals that the crosslinking manner, i.e. distance and angular flexibility between adjacent crosslinked actin filaments, is essential for the morphogenesis rather than their binding affinity and stoichiometry to actin filaments.

A statistical mechanical theory for the adsorption of protein to liposomal membranes.

The observed topology change of spherical lipid vesicles to coffee cups [Saitoh, A. et al., Proc. Natl. Acad. Sci. USA 95 (1998) 1026] was analyzed by a statistical mechanical theory. The topology change was due to the adsorption of talin molecules to the orifices of the coffee cups. The adsorption isotherm of talin between an aqueous solution and the vesicle membrane was analyzed by taking account of the bending energy of the membrane. The equilibrium is determined by the balance of the energy gain for the adsorption of talin to the periphery of the vesicles and the change of the bending energy of the membrane due to the shape change. The observed coexistence of coffee cups and sheet-like vesicles were reproduced. Vesicles with two orifices were also analyzed and theoretically reproduced.

Inhibition of microtubule assembly by HPC-1/syntaxin 1A, an exocytosis relating protein.

HPC-1/syntaxin 1A (HPC-1), which has been identified as a presynaptic membrane protein, is believed to regulate the synaptic exocytosis as a component of t-SNARE. The distribution of the protein, however, is not restricted to the synaptic terminal, but it has been found to locate on the axonal membrane. When the expression of HPC-1 was suppressed, neurite sprouting was enhanced in cultured neurons. These findings suggest that HPC-1 possesses other functions than the regulation of the membrane fusion in neurotransmitter release. Rather it may also participate in the morphogenesis of neurons through membrane fusion, and possibly through cytoskeleton. HPC-1 has a sequence resemble to the assembly promoting sequence of heat stable MAPs in residues 89-106, suggesting that it can bind tubulin and be involved in microtubule system. Thus, both the tubulin binding property and the effect on microtubule assembly of HPC-1 were examined in vitro using a mutated HPC-1 lacking the C-terminal transmembrane region (HPC-deltaTM), which was overexpressed in E. coli. Affinity column chromatography showed that tubulin was found to bind HPC-1 directly. Synthetic peptide which corresponds to the residues 89-106 competitively inhibited the tubulin-HPC-1 binding, indicating that the sequence is responsible for the tubulin binding. In addition, chemical cross-linking with EDC revealed that one HPC-1 molecule can bind per one monomeric tubulin molecule. Light scattering measurement of microtubule polymerization showed that HPC-1 decreased the rate of the pure tubulin polymerization. Direct observation of single microtubules under dark-field microscopy showed that the growth rate of microtubule decreased by HPC-1. After shortening stopped, microtubules often spent attenuate phases, in which neither growing nor shortening was detected. When another mutant HPC-1 which is composed of residues 1-97 and lacks tubulin binding activity was used, however, the suppression of microtubule polymerization was not observed. These results suggest that HPC-1 is a potent regulator of microtubule polymerization, which directly bind tubulin subunit and decrease the polymerization activity.

Microtubule-stimulated phosphorylation of tau at Ser202 and Thr205 by cdk5 decreases its microtubule nucleation activity.

Phosphorylation of tau, a heat-stable neuron-specific microtubule-associated protein, by cdk5 was stimulated in the presence of microtubules (MTs). This stimulation was due to an increased phosphorylation rate and there was no increase in total amount of phosphorylation. Two-dimensional phosphopeptide map analysis showed that MTs stimulated phosphorylation of a specific peptide. Using Western blotting with antibodies that the recognized phosphorylation-dependent epitopes within tau, the phosphorylation sites stimulated by the presence of MTs were found to be Ser202 and Thr205 (numbered according to the human tau isoform containing 441 residues). MT-dependent phosphorylation at Thr205 was observed in situ in rat cerebrum primary cultured neurons. Stimulated phosphorylation at Ser202 and Thr205 decreased the MT-nucleation activity of tau, which is in contrast to MT-independent phosphorylation at Ser235 and Ser404.

Visualization of the GDP-dependent switching in the growth polarity of microtubules.

Microtubules are filamentous polar polymers with plus and minus ends. This polarity plays a crucial role in a variety of cellular functions such as chromosome movement and organelle transport. To examine the relationship between the growth polarity of microtubules and guanine nucleotide dependence, we polymerized microtubules from axonemes of sea urchin sperm flagella either with GTP or with GTP and GDP, and observed individual microtubules by dark-field microscopy. Tubulin concentrations were adjusted in each case to grow microtubules from only one end of each axoneme. The growth polarity of microtubules was determined using N-ethylmaleimide-modified tubulin (NEM-tubulin). In the presence of GTP only and at low tubulin concentrations, microtubules grew from the plus ends of axonemes. Surprisingly, in the presence of GTP and GDP, microtubules grew from the minus ends, even at high tubulin concentrations. To confirm these results, we used a perfusion chamber to monitor the growth polarity of microtubules from the same axoneme under different conditions. Exchanging a solution containing only GTP for one containing GTP and GDP elicited a switch in the growth polarity of microtubules from the plus ends to the minus ends. These results suggest that GDP directly affects microtubule polymerization and inverts microtubule growth polarity, probably by inhibiting microtubule growth at the plus ends.

Opening-up of liposomal membranes by talin.

Morphological changes of liposomes caused by interactions between liposomal membranes and talin, a cytoskeletal submembranous protein, were studied by direct, real-time observation by using high-intensity dark-field microscopy. Surprisingly, when talin was added to a liposome solution, liposomes opened stable holes and were transformed into cup-shaped liposomes. The holes became larger with increasing talin concentration, and finally the cup-shaped liposomes were transformed into lipid bilayer sheets. These morphological changes were reversed by protein dilution, i.e., the sheets could be transformed back into closed spherical liposomes. We demonstrated that talin was localized mainly along the membrane verges, presumably avoiding exposure of its hydrophobic portion at the edge of the lipid bilayer. This is the first demonstration that a lipid bilayer can stably maintain a free verge in aqueous solution. This finding refutes the established dogma that all lipid bilayer membranes inevitably form closed vesicles and suggests that talin is a useful tool for manipulating liposomes.

Morphological transformation of liposomes caused by assembly of encapsulated tubulin and determination of shape by microtubule-associated proteins (MAPs).

To examine the role of cytoskeletons in cellular morphogenesis, we generated liposomes encapsulating tubulin, with or without microtubule-associated proteins (MAPs), and observed their transformation using dark-field microscopy. When tubulin was polymerized with MAPs in liposomes, liposomes were transformed into a "bipolar" shape with a central sphere and two tubular membrane protrusions that aligned in a straight line. On the other hand, when pure tubulin was polymerized in liposomes without MAPs, they initially transformed into a bipolar shape but subsequently re-transformed into a "monopolar" shape, i.e. a sphere with only one straight tubular portion. This re-transformation occurred in two ways: first, by shortening of one of the tubular portions due to microtubule disassembly; or second, by fluctuation of the central sphere toward one of the ends without shortening of the tube portion. MAPs prevented this re-transformation, and their role in stabilizing the shape of transformed liposomes was studied by the co-sedimentation method. The results show that MAPs, particularly MAP1 and MAP2, mediate binding between microtubules and the liposomal membrane. However, MAP2 by itself did not bind to liposomes, but was able to stabilize bipolar liposomes. This stabilization is caused not only by direct links between microtubules and liposomes, but also by prevention of Brownian motion of microtubules through an increase in friction.

Phosphorylation states of microtubule-associated protein 2 (MAP2) determine the regulatory role of MAP2 in microtubule dynamics.

Phosphorylation-dependent regulation of microtubule-stabilizing activities of microtubule-associated protein 2 (MAP2) was examined using optical microscopy. MAP2, purified from mammalian brain, was phosphorylated by either cAMP-dependent protein kinase (PKA) or cyclin B-dependent cdc2 kinase. Using PKA, 15 mol of phosphoryl groups was incorporated per mole of MAP2, but about 70% of the phosphates was distributed to the projection region. Using cdc2 kinase, 7-10 mol of phosphoryl groups was incorporated per mole of MAP2, and more than 60% of the phosphates was distributed to the microtubule-binding region. Both types of phosphorylation similarly reduced binding activity of MAP2 onto microtubules. Direct observation of individual microtubules using dark-field microscopy showed that interconversion between the polymerization phase and the depolymerization phase was repeated in both unphosphorylated and PKA-phosphorylated MAP2. In cdc2 kinase-phosphorylated MAP2, however, the phase transition from depolymerization to polymerization occurred with difficulty, with the result being that the half-life of individual microtubules was as short as in the absence of MAP2. Examination of spontaneous polymerization of microtubules using dark-field microscopy showed that the microtubule-nucleating activity of MAP2 was reduced by PKA-dependent phosphorylation and was completely abolished by cdc2 kinase-dependent phosphorylation. These observations show that cdc2 kinase-dependent phosphorylation inhibits both the microtubule-stabilizing activity and the microtubule-nucleating activity of MAP2, while PKA-dependent phosphorylation affects only the microtubule-nucleating activity of MAP2.

Self-assembly of the filament capping protein, FliD, of bacterial flagella into an annular structure.

A bacterial flagellum has a cap structure at the tip of the external filament. The cap is composed of the FliD protein (Mr, 49 x 10(3)), and plays an essential role in the polymerization of the filament protein, flagellin, which is believed to be transported through a central channel in the flagellum. A fliD-deficient mutant becomes non-motile because it lacks flagellar filaments and leaks flagellin monomer out into the medium. We have constructed a FliD-overproducing plasmid and purified the protein. The purified FliD at high concentration formed a large complex (Mr, ca. 600 x 10(3)) under physiological conditions. The complex was found by electron microscopy to be ring shaped. Image analysis revealed that the complex consisted of five substructures arranged in a pentagonal shape. Its outer diameter, approximately 10 nm, was about the same as that of the cap at the tip of the wild-type flagella. When the annular structure was added to the culture medium of a Salmonella fliD mutant, almost all of the cells became able to swim. Overall, about ten molecules of FliD self-assemble into an annular structure in vitro, forming the functional capping structure by incorporating flagellin at the tip of the flagellar filament in vivo.

Cyclin B interaction with microtubule-associated protein 4 (MAP4) targets p34cdc2 kinase to microtubules and is a potential regulator of M-phase microtubule dynamics.

We previously demonstrated (Ookata et al., 1992, 1993) that the p34cdc2/cyclin B complex associates with microtubules in the mitotic spindle and premeiotic aster in starfish oocytes, and that microtubule-associated proteins (MAPs) might be responsible for this interaction. In this study, we have investigated the mechanism by which p34cdc2 kinase associates with the microtubule cytoskeleton in primate tissue culture cells whose major MAP is known to be MAP4. Double staining of primate cells with anti-cyclin B and anti-MAP4 antibodies demonstrated these two antigens were colocalized on microtubules and copartitioned following two treatments that altered MAP4 distribution. Detergent extraction before fixation removed cyclin B as well as MAP4 from the microtubules. Depolymerization of some of the cellular microtubules with nocodazole preferentially retained the microtubule localization of both cyclin B and MAP4. The association of p34cdc2/cyclin B kinase with microtubules was also shown biochemically to be mediated by MAP4. Cosedimentation of purified p34cdc2/cyclin B with purified microtubule proteins containing MAP4, but not with MAP-free microtubules, as well as binding of MAP4 to GST-cyclin B fusion proteins, demonstrated an interaction between cyclin B and MAP4. Using recombinant MAP4 fragments, we demonstrated that the Pro-rich C-terminal region of MAP4 is sufficient to mediate the cyclin B-MAP4 interaction. Since p34cdc2/cyclin B physically associated with MAP4, we examined the ability of the kinase complex to phosphorylate MAP4. Incubation of a ternary complex of p34cdc2, cyclin B, and the COOH-terminal domain of MAP4, PA4, with ATP resulted in intracomplex phosphorylation of PA4. Finally, we tested the effects of MAP4 phosphorylation on microtubule dynamics. Phosphorylation of MAP4 by p34cdc2 kinase did not prevent its binding to microtubules, but abolished its microtubule stabilizing activity. Thus, the cyclin B/MAP4 interaction we have described may be important in targeting the mitotic kinase to appropriate cytoskeletal substrates, for the regulation of spindle assembly and dynamics.

Microtubule-stabilizing activity of microtubule-associated proteins (MAPs) is due to increase in frequency of rescue in dynamic instability: shortening length decreases with binding of MAPs onto microtubules.

The role of microtubule associated proteins (MAPs) on the dynamic instability of microtubules was examined under a dark-field microscope using bovine brain tubulin purified by DEAE-Sepharose column chromatography. In the absence of MAPs, the transition from the shortening phase to the growing phase (the rescue) occurred rarely both in self-assembled microtubules and seeded ones, especially at the plus end. Even under the conditions unfavorable to stabilize microtubule, the addition of a small amount of crude MAPs or purified microtubule associated protein 2 (MAPs) to the microtubules allowed them to undergo the rescue. At increased concentrations of MAPs or MAP2, both the length change required for a rescue during shortening phase ("shortening length") and for a catastrophe (transition from the growing to the shortening phase) ("growth length") decreased. Under these conditions, the rescue often occurred at the same site where previous rescues occurred. Distribution of immunofluorescent MAP2 antibodies along individual microtubules showed that MAP2 molecules bound onto microtubules by forming discrete clusters. The number of MAP2 molecules per cluster was estimated to be between 25 and 60. Because both the "shortening length" and the distance between MAP2 clusters in a microtubule decreased with increased MAPs concentration, we suggest that the MAP2 clusters may form the specific site at which the shortening of the microtubule readily stops. MAP2 possibly regulates the dynamic instability by stopping the shortening, which is a prerequisite for the rescue.

Movement of the F40 domain of flagellin during the morphological transition of bacterial flagella.

Flagella from Salmonella typhimurium were labeled with various amounts of fluorescein isothiocyanate. The site of labeling was identified as being predominantly in the exterior F40 domain. The fluorescence intensity decreased as the fluorescein density on the flagella increased, indicating self energy transfer between fluoresceins. The fluorescence of modified flagella was measured during the normal-to-curly morphological transition induced by alkaline pH. The morphological transition itself was simultaneously monitored by dark-field microscopy. Concomitant with the transition was a 25% increase in fluorescence for flagella heavily labeled with fluorescein. This was shown to be due to a decrease in the efficiency of energy transfer between fluoresceins on proximal flagellin subunits, implying that the F40 domains undergo relative movement apart during the morphological transition. Closer inspection of the domain movement and morphological transition as a function of pH reveals that the two processes are not exactly concomitant. This indicates the existence of intermediates during the transition. The fluorescence technique, outlined here, provides a means of directly monitoring an organizational 'switch' in the flagellin subunits during the actual morphological transition of flagella.

The effect of sugars on the morphology of the bacterial flagellum.

Using dark-field microscopy, we have found that certain sugars cause the normal-to-curly helical transition of bacterial flagella. Titration of flagella isolated from Salmonella typhimurium with 16 different carbohydrates showed that: (i) only certain sugars cause the transition. There is no obvious relationship between the simple physico-chemical properties of the sugar and whether the sugar causes the transition or not; (ii) the efficacies of sugars that do cause the transition differ markedly. For these sugars there is a relationship between efficacy and molecular size. These results suggest that the specific, though weak, binding of sugars to sites on flagella cause the morphological transition.

Flagellar growth in a filament-less Salmonella fliD mutant supplemented with purified hook-associated protein 2.

Bacterial flagellum consists of a basal body, a hook, HAP1 (hook-associated protein 1), HAP3, a long helical filament, and a cap (composed of HAP2), all connected in series. The mutant deficient in the HAP2 structural gene (fliD) of Salmonella typhimurium has flagella composed of only hook-HAP1-HAP3 and excretes flagellin monomers into the culture medium. However, when purified HAP2 was added to this mutant, the flagellin stopped leaking out and flagellar filaments grew. Turnover of HAP2 was not necessary for the growth of a filament. Therefore HAP2 facilitates the polymerization of endogenous flagellin, apparently without falling off the filament tip. This experimental system with exogenous HAP2 allowed us to synchronize filament growth; the average rate of filament growth can be estimated by measuring the length of grown filaments at various time periods in electron micrographs. The initial growth rate was about 30 nm/min, which corresponds to one flagellin per second.