Tomomi Tani

Unc-51-like kinase 1/2-mediated endocytic processes regulate filopodia extension and branching of sensory axons

The molecular mechanism and significance of endocytic processes involved in directional axon elongation are not well understood. The Unc-51 family of serine/threonine kinases was shown to be important for axon growth and was also linked to endocytosis, providing an entry point to study this problem. We found that mouse Unc-51-like kinase 1/2 (Ulk1/2) proteins are localized to vesicular structures in growth cones of mouse spinal sensory neurons. RNAi-mediated knockdown of Ulk1 and/or Ulk2 resulted in impaired endocytosis of nerve growth factor (NGF), excessive axon arborization, and severely stunted axon elongation. The evidence also indicates that Ulk1/2 mediates a non-clathrin-coated endocytosis in sensory growth cones. Interestingly, NGF can induce the interaction of Ulk1 with TrkA receptor complexes through promoting K63-polyubiquitination of Ulk1 and binding of Ulk1 to the scaffolding protein p62. These results and additional studies suggest that Ulk1/2 proteins regulate filopodia extension and neurite branching during sensory axon outgrowth, probably through regulating TrkA receptor trafficking and signaling.

An ultramarine fluorescent protein with increased photostability and pH insensitivity

We report a pH-insensitive and photostable ultramarine fluorescent protein, Sirius, with an emission peak at 424 nm, the shortest emission wavelength among fluorescent proteins reported to date. The pH-insensitivity of Sirius allowed prolonged visualization of biological events in an acidic environment. Two fluorescence resonance energy transfer (FRET) pairs, Sirius-mseCFP and Sapphire-DsRed, allowed dual-FRET imaging with single-wavelength excitation, enabling detection of Ca2+ concentration and caspase-3 activation in the same apoptotic cells.

Septin assemblies form by diffusion-driven annealing on membranes

Significance The mechanisms and location of polymerization and disassembly direct the function of cytoskeletal proteins. Septins are far less understood than other cytoskeletal elements such as actin and microtubules, yet they have a conserved function acting as scaffolds at cell membranes and are implicated in cancers, neurodegenerative diseases, and microbial pathogenesis. We have defined a key role of the membrane in directing septin filament formation in live cells and reconstituted dynamic septin polymerization, using purified components. We find that septins grow into filaments and form higher-order structures by diffusing, colliding, and annealing on the plasma membrane. This work is important because it defines previously unidentified basic steps of polymerization and construction of higher-order assemblies of septin proteins. Septins assemble into filaments and higher-order structures that act as scaffolds for diverse cell functions including cytokinesis, cell polarity, and membrane remodeling. Despite their conserved role in cell organization, little is known about how septin filaments elongate and are knitted together into higher-order assemblies. Using fluorescence correlation spectroscopy, we determined that cytosolic septins are in small complexes, suggesting that septin filaments are not formed in the cytosol. When the plasma membrane of live cells is monitored by total internal reflection fluorescence microscopy, we see that septin complexes of variable size diffuse in two dimensions. Diffusing septin complexes collide and make end-on associations to form elongated filaments and higher-order structures, an assembly process we call annealing. Septin assembly by annealing can be reconstituted in vitro on supported lipid bilayers with purified septin complexes. Using the reconstitution assay, we show that septin filaments are highly flexible, grow only from free filament ends, and do not exchange subunits in the middle of filaments. This work shows that annealing is a previously unidentified intrinsic property of septins in the presence of membranes and demonstrates that cells exploit this mechanism to build large septin assemblies.

Local Nucleosome Dynamics Facilitate Chromatin Accessibility in Living Mammalian Cells

Genome information, which is three-dimensionally organized within cells as chromatin, is searched and read by various proteins for diverse cell functions. Although how the protein factors find their targets remains unclear, the dynamic and flexible nature of chromatin is likely crucial. Using a combined approach of fluorescence correlation spectroscopy, single-nucleosome imaging, and Monte Carlo computer simulations, we demonstrate local chromatin dynamics in living mammalian cells. We show that similar to interphase chromatin, dense mitotic chromosomes also have considerable chromatin accessibility. For both interphase and mitotic chromatin, we observed local fluctuation of individual nucleosomes (~50 nm movement/30 ms), which is caused by confined Brownian motion. Inhibition of these local dynamics by crosslinking impaired accessibility in the dense chromatin regions. Our findings show that local nucleosome dynamics drive chromatin accessibility. We propose that this local nucleosome fluctuation is the basis for scanning genome information.

Trafficking of a Ligand-Receptor Complex on the Growth Cones as an Essential Step for the Uptake of Nerve Growth Factor at the Distal End of the Axon: A Single-Molecule Analysis

The behavior of single molecules of neurotrophins on growth cones was observed by the use of the fluorescent conjugate of nerve growth factor (NGF), Cy3-NGF. After the application of 0.4 nm Cy3-NGF, chick dorsal root ganglion growth cones responded within 1 min of adding the stimulus by expanding their lamellipodia. Only 40 molecules of Cy3-NGF, which occupied <5% of the estimated total binding sites on a single growth cone, were required to initiate the motile responses. After binding to the high-affinity receptor, Cy3-NGF displayed lateral diffusion on the membrane of the growth cones with a diffusion constant of 0.3 μm2 s-1. The behavior of Cy3-NGF was shifted to a one-directional rearward movement toward the central region of the growth cone. The one-directional movement of Cy3-NGF displayed the same rate as the rearward flow of actin, ∼4 μm/min. This movement could be stopped by the application of the potent inhibitor of actin polymerization, latrunculin B. Molecules of Cy3-NGF were suggested to be internalized in the vicinity of the central region of the growth cone during this rearward trafficking, because Cy3-NGF remained in the growth cone after the growth cones had been exposed to an acidic surrounding medium: acidic medium causes the complete dissociation of Cy3-NGF from the receptors on the surface of growth cones. These results suggested that actin-driven trafficking of the NGF receptor complex is an essential step for the accumulation and endocytosis of NGF at the growth cone and for the retrograde transport of NGF toward the cell body.

Family 3 β-glucosidase from cellulose-degrading culture of the white-rot fungus Phanerochaete chrysosporium is a glucan 1,3-β-glucosidase

Abstract The substrate specificity of an extracellular β-glucosidase (BGL) from cellulose-degrading culture of the white-rot fungus Phanerochaete chrysosporium was investigated, using a variety of compounds with β-glucosidic linkages. Amino acid sequencing data for the purified BGL showed that the enzyme is identical to the glycoside hydrolase (GH) family 3 BGL of the same fungus previously reported [Li, B. and Renganathan, V, Appl. Environ. Microbiol., 64, 2748-2754 (1998)]. The BGL can hydrolyze both cellobiose and cellobionolactone, but cellobionolactone was hydrolyzed very much more slowly than cellobiose. Moreover, cellobionolactone inhibited cellobiose hydrolysis by the BGL, suggesting that this enzyme cannot cooperate with cellobiose dehydrogenase (CDH) in cellulose degradation by P. chrysosporium. In addition to cellobiose, BGL utilized various glucosyl-β-glucosides, such as sophorose, laminaribiose and gentiobiose, as substrates. Among the four substrates, laminaribiose (β-1,3-glucosidic linkage) was hydrolyzed most effectively. Moreover, the hydrolytic rate of laminarioligosaccharides increased proportionally to the degree of polymerization (DP), and the activity of BGL even towards laminarin with an average DP of 25 was similar to that towards laminaripentaose (DP 5). Therefore, we conclude that the extracellular BGL from P. chrysosporium is primarily a glucan 1,3-β-glucosidase (EC 3.2.1.58), which might play a role on fungal cell wall metabolism, rather than a β-glucosidase (EC 3.2.1.21), which might be involved in the hydrolysis of β-1,4-glucosidic compounds during cellulose degradation.

Reconstitution of dynamic microtubules with Drosophila XMAP215, EB1, and Sentin

XMAP215msps and the EB1–Sentin duo act individually and cooperatively to accelerate microtubule growth and increase rescue events but also to promote frequent catastrophes.

Particle Tracking of Intracellular Trafficking of Octaarginine-modified Liposomes: A Comparative Study With Adenovirus

It is previously reported that octaarginine (R8)-modified liposome (R8-Lip) was taken up via macropinocytosis, and subsequently delivered to the nuclear periphery. In the present study, we investigated the mechanism for the cytoplasmic transport of R8-Lips, comparing with that for adenovirus. Treatment with microtubule-disruption reagent (nocodazole) inhibited the transfection activity of plasmid DNA (pDNA)-encapsulating R8-Lip more extensively than that of adenovirus. The directional transport of R8-Lips along green fluorescent protein (GFP)-tagged microtubules was observed; however, the velocity was slower than those for adenovirus or endosomes that were devoid of R8-Lips. These directional motions were abrogated in R8-Lips by nocodazole treatment, whereas adenovirus continued to undergo random motion. This finding suggests that the nuclear access of R8-Lip predominantly involves microtubule-dependent transport, whereas an apparent diffusive motion is also operative in nuclear access of adenovirus. Furthermore, quantum dot-labeled pDNA underwent directional motion concomitantly with rhodamine-labeled lipid envelopes, indicating that the R8-Lips were subject to microtubule-dependent transport in the intact form. Dual particle tracking of carriers and endosomes revealed that R8-Lip was directionally transported, associated with endosomes, whereas this occurs after endosomal escape in adenovirus. Collectively, the findings reported herein indicate that vesicular transport is a key factor in the cytoplasmic transport of R8-Lips.

Flexible and dynamic nucleosome fiber in living mammalian cells.

Genomic DNA is organized three dimensionally within cells as chromatin and is searched and read by various proteins by an unknown mechanism; this mediates diverse cell functions. Recently, several pieces of evidence, including our cryomicroscopy and synchrotron X-ray scattering analyses, have demonstrated that chromatin consists of irregularly folded nucleosome fibers without a 30-nm chromatin fiber (i.e., a polymer melt-like structure). This melt-like structure implies a less physically constrained and locally more dynamic state, which may be crucial for protein factors to scan genomic DNA. Using a combined approach of fluorescence correlation spectroscopy, Monte Carlo computer simulations, and single nucleosome imaging, we demonstrated the flexible and dynamic nature of the nucleosome fiber in living mammalian cells. We observed local nucleosome fluctuation (~50 nm movement per 30 ms) caused by Brownian motion. Our in vivo-in silico results suggest that local nucleosome dynamics facilitate chromatin accessibility and play a critical role in the scanning of genome information.

Cytoplasmic Nucleation and Atypical Branching Nucleation Generate Endoplasmic Microtubules in Physcomitrella patens

Unconventional modes of microtubule nucleation exist in the moss cytoplasm, representing a broader spectrum of nucleation mechanisms in plants. The mechanism underlying microtubule (MT) generation in plants has been primarily studied using the cortical MT array, in which fixed-angled branching nucleation and katanin-dependent MT severing predominate. However, little is known about MT generation in the endoplasm. Here, we explored the mechanism of endoplasmic MT generation in protonemal cells of Physcomitrella patens. We developed an assay that utilizes flow cell and oblique illumination fluorescence microscopy, which allowed visualization and quantification of individual MT dynamics. MT severing was infrequently observed, and disruption of katanin did not severely affect MT generation. Branching nucleation was observed, but it showed markedly variable branch angles and was occasionally accompanied by the transport of nucleated MTs. Cytoplasmic nucleation at seemingly random locations was most frequently observed and predominated when depolymerized MTs were regrown. The MT nucleator γ-tubulin was detected at the majority of the nucleation sites, at which a single MT was generated in random directions. When γ-tubulin was knocked down, MT generation was significantly delayed in the regrowth assay. However, nucleation occurred at a normal frequency in steady state, suggesting the presence of a γ-tubulin-independent backup mechanism. Thus, endoplasmic MTs in this cell type are generated in a less ordered manner, showing a broader spectrum of nucleation mechanisms in plants.