Using Imaging Flow Cytometry to Quantify and Optimize Giant Vesicle Production by Water-in-oil Emulsion Transfer Methods.

Many biologists, biochemists, and biophysicists study giant vesicles, which have a diameter of >1 μm, owing to their ease of characterization using standard optical methods. More recently, there has been interest in using giant vesicles as model systems for living cells and for the construction of artificial cells. In fact, there have been a number of reports about functionalizing giant vesicles using membrane-bound pore proteins and encapsulating biochemical reactions.

Near-infrared light-directed RNAi using a photosensitive carrier molecule.

Controlled activation of small RNAs, such as small interfering RNA, in cells is very useful for various biological applications. Light is an effective inducer of controlled activation; in particular, near-infrared light is favorable because it can penetrate deeper into tissues than UV or visible light. In this study, near-infrared light control of RNA interference (RNAi) was demonstrated in mammalian cells using a photosensitive RNA carrier molecule, consisting of an RNA carrier protein and a fluorochrome.

The C-terminal α-helix of SPAS-1, a Caenorhabditis elegans spastin homologue, is crucial for microtubule severing.

Spastin belongs to the meiotic subfamily, together with Vps4/SKD1, fidgetin and katanin, of AAA (ATPases associated with diverse cellular activities) proteins, and functions in microtubule severing. Interestingly, all members of this subgroup specifically contain an additional α-helix at the very C-terminal end. To understand the function of the C-terminal α-helix, we characterised its deletion mutants of SPAS-1, a Caenorhabditis elegans spastin homologue, in vitro and in vivo.

Photoinduced RNA interference.

Because RNA interference (RNAi) can be applied to any gene, this technique has been widely used for studying gene functions. In addition, many researchers are attempting to use RNAi technology in RNAi-based therapies. However, several challenging and controversial issues have arisen during the widespread application of RNAi including target gene specificity, target cell specificity, and spatiotemporal control of gene silencing.

Photosensitizing carrier proteins for photoinducible RNA interference.

RNA interference (RNAi) is being widely explored as a tool in functional genomics and tissue engineering, and in the therapy of intractable diseases, including cancer and neurodegenerative diseases. Recently, we developed a photoinducible RNAi method using photosensitizing carrier proteins, named CLIP-RNAi (CPP-linked RBP-mediated RNA internalization and photoinduced RNAi). Novel carrier proteins were designed for this study to establish a highly efficient delivery system for small interfering RNA (siRNA) or short hairpin RNA (shRNA) and to demonstrate light-dependent gene silencing.

Conserved aromatic and basic amino acid residues in the pore region of Caenorhabditis elegans spastin play critical roles in microtubule severing.

Mutations of human spastin, an AAA (ATPases associated with diverse cellular activity) family protein, cause an autosomal dominant form of hereditary spastic paraplegia, which is characterized by weakness, spasticity and loss of the vibratory sense in the lower limbs. Recently, it has been reported that spastin displays microtubule-severing activity. We also previously reported that Caenorhabditis elegans spastin homologue SPAS-1 displays microtubule severing.

From the common molecular basis of the AAA protein to various energy-dependent and -independent activities of AAA proteins.

AAA (ATPase associated with various cellular activities) proteins remodel substrate proteins and protein complexes upon ATP hydrolysis. Substrate remodelling is diverse, e.g.

The C. elegans homologue of the spastic paraplegia protein, spastin, disassembles microtubules.

Mutations in human spastin (SPG4) cause an autosomal dominant form of hereditary spastic paraplegia. Sequence analysis revealed that spastin contains the AAA (ATPases associated with diverse cellular activities) domain in the C-terminal region. Recently, it was reported that spastin interacts dynamically with microtubules and displays microtubule-severing activity.