Yohko Tanaka-Takiguchi

Septin-Mediated Uniform Bracing of Phospholipid Membranes

Cell shape is determined by the interplay between the lipid bilayer and the underlying network of protein polymers. We explored unknown determinants involved in cell morphogenesis as factors that transform phospholipid-based liposomes (diameter 5-20 microm). Unlabeled giant liposomes, observed through dark-field optics, were metastable in an aqueous suspension. In contrast, liposomes robustly protruded uniform tubules immediately after the addition of a brain extract to the suspension. The tubulation reaction was greatly facilitated when the liposomes contained PIP or PIP2. Biochemical analysis of the brain extract revealed that heteromeric complexes of septins, a family of polymerizing GTP/GDP-binding proteins, are responsible for the membrane transformation. Ultrastructural analysis established that each membrane tubule (diameter 0.43 +/- 0.079 microm) is braced by a circumferential array of septin filaments. Although submembranous septin assemblies are associated with diverse cortical morphogenesis from yeast to mammals, the biophysical basis for the septin-membrane interplay remains largely unknown. Further, there is a biochemical discrepancy between the fast septin remodeling in cells and their slow self-assembly in vitro. This membrane-facilitated fast septin assembly demonstrated for the first time by our unique experimental system should provide important clues to characterize these processes.

Entrapping desired amounts of actin filaments and molecular motor proteins in giant liposomes.

We have successfully prepared cell-sized giant liposomes encapsulating desired amounts of actoHMM, a mixture of actin filament (F-actin) and heavy meromyosin (HMM, an actin-related molecular motor), in the presence of 5 mM MgCl 2 and 50 mM KCl. We employed a spontaneous transfer method to prepare those liposomes. In the absence of HMM, F-actin was distributed homogeneously inside the liposomes. In contrast, when F-actin was encapsulated in liposomes together with HMM, network structures were generated. Such network structures are attributable to the cross-linking of F-actin by HMM.

Transformation of ActoHMM Assembly Confined in Cell-Sized Liposome

To construct a simple model of a cellular system equipped with motor proteins, cell-sized giant liposomes encapsulating various amounts of actoHMM, the complexes of actin filaments (F-actin) and heavy meromyosin (HMM, an actin-related molecular motor), with a depletion reagent to mimic the crowding effect of inside of living cell, were prepared. We adapted the methodology of the spontaneous transfer of water-in-oil (W/O) droplets through a phospholipid monolayer into the bulk aqueous phase and successfully prepared stable giant liposomes encapsulating the solution with a physiological salt concentration containing the desired concentrations of actoHMM, which had been almost impossible to obtain using currently adapted methodologies such as natural swelling and electro-formation on an electrode. We then examined the effect of ATP on the cytoskeleton components confined in those cell-sized liposomes, because ATP is known to drive the sliding motion for actoHMM. We added α-hemolysin, a bacterial membrane pore-forming toxin, to the bathing solution and obtained liposomes with the protein pores embedded on the bilayer membrane to allow the transfer of ATP inside the liposomes. We show that, by the ATP supply, the actoHMM bundles inside the liposomes exhibit specific changes in spatial distribution, caused by the active sliding between F-actin and HMM. Interestingly, all F-actins localized around the inner periphery of liposomes smaller than a critical size, whereas in the bulk solution and also in larger liposomes, the actin bundles formed aster-like structures under the same conditions.

Physicochemical analysis from real-time imaging of liposome tubulation reveals the characteristics of individual F-BAR domain proteins.

The Fer-CIP4 homology-BAR (F-BAR) domain, which was identified as a biological membrane-deforming module, has been reported to transform lipid bilayer membranes into tubules. However, details of the tubulation process, the mechanism, and the properties of the generated tubules remain unknown. Here, we successfully monitored the entire process of tubulation and the behavior of elongated tubules caused by four different F-BAR domain family proteins (FBP17, CIP4, PSTPIP1, and Pacsin2) using direct real-time imaging of giant unilamellar liposomes with dark-field optical microscopy. FBP17 and CIP4 develop many protrusions simultaneously over the entire surface of individual liposomes, whereas PSTPIP1 and Pacsin2 develop only a few protrusions from a narrow restricted part of the surface of individual liposomes. Tubules formed by FBP17 or CIP4 have higher bending rigidities than those formed by PSTPIP1 or Pacsin2. The results provide striking evidence that these four F-BAR domain family proteins should be classified into two groups: one group of FBP17 and CIP4 and another group of PSTPIP1 and Pacsin2. This classification is consistent with the phylogenetic proximity among these proteins and suggests that the nature of the respective tubulation is associated with biological function. These findings aid in the quantitative assessment with respect to manipulating the morphology of lipid bilayers using membrane-deforming proteins.

Multiple Membrane Interactions and Versatile Vesicle Deformations Elicited by Melittin

Melittin induces various reactions in membranes and has been widely studied as a model for membrane-interacting peptide; however, the mechanism whereby melittin elicits its effects remains unclear. Here, we observed melittin-induced changes in individual giant liposomes using direct real-time imaging by dark-field optical microscopy, and the mechanisms involved were correlated with results obtained using circular dichroism, cosedimentation, fluorescence quenching of tryptophan residues, and electron microscopy. Depending on the concentration of negatively charged phospholipids in the membrane and the molecular ratio between lipid and melittin, melittin induced the “increasing membrane area”, “phased shrinkage”, or “solubilization” of liposomes. In phased shrinkage, liposomes formed small particles on their surface and rapidly decreased in size. Under conditions in which the increasing membrane area, phased shrinkage, or solubilization were mainly observed, the secondary structure of melittin was primarily estimated as an α-helix, β-like, or disordered structure, respectively. When the increasing membrane area or phased shrinkage occurred, almost all melittin was bound to the membranes and reached more hydrophobic regions of the membranes than when solubilization occurred. These results indicate that the various effects of melittin result from its ability to adopt various structures and membrane-binding states depending on the conditions.

Construction of Cell-Sized Liposomes Encapsulating Actin and Actin-Cross-linking Proteins

To shed light on the mechanism underlying the active morphogenesis of living cells in relation to the organization of internal cytoskeletal networks, the development of new methodologies to construct artificial cell models is crucial. Here, we describe the successful construction of cell-sized liposomes entrapping cytoskeletal proteins. We discuss experimental protocols to prepare giant liposomes encapsulating desired amounts of actin and cross-linking proteins including molecular motor proteins, such as fascin, alpha-actinin, filamin, myosin-I isolated from brush border (BBMI), and heavy meromyosin (HMM). Subfragment 1 (S-1) is also studied in comparison to HMM, where S-1 and HMM are single-headed and double-headed derivatives of conventional myosin (myosin-II), respectively. In the absence of cross-linking proteins, actin filaments (F-actin) are distributed homogeneously without any order within the liposomes. In contrast, when actin is encapsulated together with an actin-cross-linking protein, mesh structures emerge that are similar to those in living motile cells. Optical microscopic observations on the active morphological changes of the liposomes are reported.

Multiple kinesin-14 family members drive microtubule minus end–directed transport in plant cells

Minus end–directed cargo transport along microtubules (MTs) is exclusively driven by the molecular motor dynein in a wide variety of cell types. Interestingly, during evolution, plants have lost the genes encoding dynein; the MT motors that compensate for dynein function are unknown. Here, we show that two members of the kinesin-14 family drive minus end–directed transport in plants. Gene knockout analyses of the moss Physcomitrella patens revealed that the plant-specific class VI kinesin-14, KCBP, is required for minus end–directed transport of the nucleus and chloroplasts. Purified KCBP directly bound to acidic phospholipids and unidirectionally transported phospholipid liposomes along MTs in vitro. Thus, minus end–directed transport of membranous cargoes might be driven by their direct interaction with this motor protein. Newly nucleated cytoplasmic MTs represent another known cargo exhibiting minus end–directed motility, and we identified the conserved class I kinesin-14 (ATK) as the motor involved. These results suggest that kinesin-14 motors were duplicated and developed as alternative MT-based minus end–directed transporters in land plants.

Pico-liter injection control to individual nano-liter solution coated by lipid layer

This paper presents the evaluation of ultra-minimal spout amount from micro-nano pipettes into phospholipid-coated micro-droplets. The pipettes can be used to control the local environment around/inside single cells. Conventionally, the microinjection with pipette has been conducted by air pressures. In this method, the spout of high viscosity solutions is difficult because of the frictional forces between the surface of a pipette and a solution. It is also needed to evaluate the spout amount quantitatively. On the other hand, the research about artificial cell model has been actively conducted, injecting various biological samples into liposomes which are vesicle of lipid bilayer membrane. If additional injection of various proteins into liposome is realized with micro/nano pipette, the observation of dynamic reaction of multiple biological samples, which is required for formulation of artificial cell, will become possible. In our research, the spouting method using electro-osmosis is used. The local spouts of the proteins such as GFP and F-actin have been presented experimentally before. In this paper, the amount of spout solution in this method is quantitatively evaluated by injection of fluorescent solution into a phospholipid-coated micro-droplet.

Specific Transformation of Assembly with Actin Filaments and Molecular Motors in a Cell-Sized Self-Emerged Liposome

Eukaryotes, by the same combination of cytoskeleton and molecular motor, for example actin filament and myosin, can generate a variety of movements. For this diversity, the organization of biological machineries caused by the confinement and/or crowding effects of internal living cells, may play very important roles.

Reconstruction of motile actin networks in giant liposome

To construct a simple model cellular system exhibiting the property of self-propelled motion, cell-sized giant liposomes encapsulating desired amounts of actoHMM, a mixture of actin filament (F-actin) and heavy meromyosin (HMM, an actin-related molecular motor), have been prepared. We adapted the methodology of spontaneous transfer of a water droplet through oil/water interface in the presence of phospholipid and successful obtained stable giant liposome with the inner physiological biopolymer solution. We introduced ATP to the bathing solution of liposome encapsulating actoHMM, in which bilayer membrane α-hemolysin, a bacterial membrane pore-forming toxin, is embedded. In this system, ATP is supplied into the inner volume of liposome through the protein pores in a passive manner. Accompanied by the ATP supply, actin networks or bundles that have encapsulated in the liposomes exhibited specific morphological change, being attributable to the active sliding between F-actin and HMM. Remarkable difference in the behavior of F-actins is found; i.e., inside the liposome, almost all the F-actins situate around the inner periphery of the liposome, whereas, in the bulk solution, actin bundles form an aster-like structure.