Single Quantum Dot Tracking Reveals that an Individual Multivalent HIV-1 Tat Protein Transduction Domain Can Activate Machinery for Lateral Transport and Endocytosis

The mechanisms underlying the cellular entry of the HIV-1 Tat protein transduction domain (TatP) and the molecular information necessary to improve the transduction efficiency of TatP remain unclear due to the technical limitations for direct visualization of TatP''s behavior in cells. Using confocal microscopy, total internal reflection fluorescence microscopy, and four-dimensional microscopy, we developed a single-molecule tracking assay for TatP labeled with quantum dots (QDs) to examine the kinetics of TatP initially and immediately before, at the beginning of, and immediately after entry into living cells. We report that even when the number of multivalent TatP (mTatP)-QDs bound to a cell was low, each single mTatP-QD first locally induced the cell''s lateral transport machinery to move the mTatP-QD toward the center of the cell body upon cross-linking of heparan sulfate proteoglycans. The centripetal and lateral movements were linked to the integrity and flow of actomyosin and microtubules. Individual mTatP underwent lipid raft-mediated temporal confinement, followed by complete immobilization, which ultimately led to endocytotic internalization. However, bivalent TatP did not sufficiently promote either cell surface movement or internalization. Together, these findings provide clues regarding the mechanisms of TatP cell entry and indicate that increasing the valence of TatP on nanoparticles allows them to behave as cargo delivery nanomachines.     

Focus Issue on Calcium Signaling: Analyses of a Gravistimulation-Specific Ca2+ Signature in Arabidopsis using Parabolic Flights

Gravity is a critical environmental factor affecting the morphology and functions of organisms on the Earth. Plants sense changes in the gravity vector (gravistimulation) and regulate their growth direction accordingly. In Arabidopsis (Arabidopsis thaliana) seedlings, gravistimulation, achieved by rotating the specimens under the ambient 1g of the Earth, is known to induce a biphasic (transient and sustained) increase in cytoplasmic calcium concentration ([Ca2+]_c). However, the [Ca2+]_c increase genuinely caused by gravistimulation has not been identified because gravistimulation is generally accompanied by rotation of specimens on the ground (1g), adding an additional mechanical signal to the treatment. Here, we demonstrate a gravistimulation-specific Ca²⁺ response in Arabidopsis seedlings by separating rotation from gravistimulation by using the microgravity (less than 10⁻⁴g) conditions provided by parabolic flights. Gravistimulation without rotating the specimen caused a sustained [Ca2+]_c increase, which corresponds closely to the second sustained [Ca2+]_c increase observed in ground experiments. The [Ca2+]_c increases were analyzed under a variety of gravity intensities (e.g. 0.5g, 1.5g, or 2g) combined with rapid switching between hypergravity and microgravity, demonstrating that Arabidopsis seedlings possess a very rapid gravity-sensing mechanism linearly transducing a wide range of gravitational changes (0.5g–2g) into Ca²⁺ signals on a subsecond time scale.Calcium ion (Ca²⁺) functions as an intracellular second messenger in many signaling pathways in plants (White and Broadley, 2003; Hetherington and Brownlee, 2004; McAinsh and Pittman, 2009; Spalding and Harper, 2011). Endogenous and exogenous signals are spatiotemporally encoded by changing the free cytoplasmic concentration of Ca²⁺ ([Ca2+]_c), which in turn triggers [Ca2+]_c-dependent downstream signaling (Sanders et al., 2002; Dodd et al., 2010). A variety of [Ca2+]_c increases induced by diverse environmental and developmental stimuli are reported, such as phytohormones (Allen et al., 2000), temperature (Plieth et al., 1999; Dodd et al., 2006), and touch (Knight et al., 1991; Monshausen et al., 2009). The [Ca2+]_c increase couples each stimulus and appropriate physiological responses. In the Ca²⁺ signaling pathways, the stimulus-specific [Ca2+]_c pattern (e.g. amplitude and oscillation) provide the critical information for cellular signaling (Scrase-Field and Knight, 2003; Dodd et al., 2010). Therefore, identification of the stimulus-specific [Ca2+]_c signature is crucial for an understanding of the intracellular signaling pathways and physiological responses triggered by each stimulus, as shown in the case of cold acclimation (Knight et al., 1996; Knight and Knight, 2000).Plants often exhibit biphasic [Ca2+]_c increases in response to environmental stimuli. Thus, slow cooling causes a fast [Ca2+]_c transient followed by a second, extended [Ca2+]_c increase in Arabidopsis (Arabidopsis thaliana; Plieth et al., 1999; Knight and Knight, 2000). The Ca²⁺ channel blocker lanthanum (La³⁺) attenuated the fast transient but not the following increase (Knight and Knight, 2000), suggesting that these two [Ca2+]_c peaks have different origins. Similarly, hypoosmotic shock caused a biphasic [Ca2+]_c increase in tobacco (Nicotiana tabacum) suspension-culture cells (Takahashi et al., 1997; Cessna et al., 1998). The first [Ca2+]_c peak was inhibited by gadolinium (Gd³⁺), La³⁺, and the Ca²⁺ chelator EGTA (Takahashi et al., 1997; Cessna et al., 1998), whereas the second [Ca2+]_c increase was inhibited by the intracellular Ca²⁺ store-depleting agent caffeine but not by EGTA (Cessna et al., 1998). The amplitude of the first [Ca2+]_c peak affected the amplitude of the second increase and vice versa (Cessna et al., 1998). These results suggest that even though the two [Ca2+]_c peaks originate from different Ca²⁺ fluxes (e.g. Ca²⁺ influx through the plasma membrane and Ca²⁺ release from subcellular stores, respectively), they are closely interrelated, showing the importance of the kinetic and pharmacological analyses of these [Ca2+]_c increases.Changes in the gravity vector (gravistimulation) could work as crucial environmental stimuli in plants and are generally achieved by rotating the specimens (e.g. +180°) in ground experiments. Use of Arabidopsis seedlings expressing apoaequorin, a Ca²⁺-reporting photoprotein (Plieth and Trewavas, 2002; Toyota et al., 2008a), has revealed that gravistimulation induces a biphasic [Ca2+]_c increase that may be involved in the sensory pathway for gravity perception/response (Pickard, 2007; Toyota and Gilroy, 2013) and the intracellular distribution of auxin transporters (Benjamins et al., 2003; Zhang et al., 2011). These two Ca²⁺ changes have different characteristics. The first transient [Ca2+]_c increase depends on the rotational velocity but not angle, whereas the second sustained [Ca2+]_c increase depends on the rotational angle but not velocity. The first [Ca2+]_c transient was inhibited by Gd³⁺, La³⁺, and the Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid but not by ruthenium red (RR), whereas the second sustained [Ca2+]_c increase was inhibited by all these chemicals. These results suggest that the first transient and second sustained [Ca2+]_c increases are related to the rotational stimulation and the gravistimulation, respectively, and are mediated by distinct molecular mechanisms (Toyota et al., 2008a). However, it has not been demonstrated directly that the second sustained [Ca2+]_c increase is induced solely by gravistimulation; it could be influenced by other factors, such as an interaction with the first transient [Ca2+]_c increase (Cessna et al., 1998), vibration, and/or deformation of plants during the rotation.To elucidate the genuine Ca²⁺ signature in response to gravistimulation in plants, we separated rotation and gravistimulation under microgravity (μg; less than 10⁻⁴g) conditions provided by parabolic flight (PF). Using this approach, we were able to apply rotation and gravistimulation to plants separately (Fig. 1). When Arabidopsis seedlings were rotated +180° under μg conditions, the [Ca2+]_c response to the rotation was transient and almost totally attenuated in a few seconds. Gravistimulation (transition from μg to 1.5g) was then applied to these prerotated specimens at the terminating phase of the PF. This gravistimulation without simultaneous rotation induced a sustained [Ca2+]_c increase. The kinetic properties of this sustained [Ca2+]_c increase were examined under different gravity intensities (0.5g–2g) and sequences of gravity intensity changes (Fig. 2A). This analysis revealed that gravistimulation-specific Ca²⁺ response has an almost linear dependency on gravitational acceleration (0.5g–2g) and an extremely rapid responsiveness of less than 1 s.Open in a separate windowFigure 1.Diagram of the experimental procedures for applying separately rotation and gravistimulation to Arabidopsis seedlings. Rotatory stimulation (green arrow) was applied by rotating the seedlings 180° under μg conditions, and 1.5g 180° rotation gravistimulation (blue arrow) was applied to the prerotated seedlings after μg.Open in a separate windowFigure 2.Acceleration, temperature, humidity, and pressure in an aircraft during flight experiments. A, Accelerations along x, y, and z axes in the aircraft during PF. The direction of flight (FWD) and coordinates (x, y, and z) are indicated in the bottom graph. The inset shows an enlargement of the acceleration along the z axis (gravitational acceleration) during μg conditions lasting for approximately 20 s. B, Temperature, humidity, and pressure in the aircraft during PF. Shaded areas in graphs denote the μg condition.     

Estimation of the fertility rates of Japanese wild boars (Sus scrofa leucomystax) using fetuses and corpora albicans

Effective population control of Japanese wild boar (Sus scrofa leucomystax) requires reliable information about population dynamics. Fertility rate is the fundamental component of reproduction to evaluate population dynamics. However, little is known regarding the fertility rate of Japanese wild boar. The traditional hunting practices make it difficult to obtain pregnant females and calculate the fertility rate by checking fetuses as is performed in other countries. Therefore, we focused on the corpora albicans (CA) as the CA remains in the ovaries of postpartum females after pregnancy. This study aimed to evaluate the utility of CA and estimate the fertility rate of Japanese wild boars using CA. Histological analysis of ovaries enabled us to discriminate type 1 CA, which remains for 1 year after breeding. Type 1 CA is a superior indicator compared with lactation in the non-pregnancy season because it allows verification of postpartum females over a long period. The fertility rate was calculated by the combination of pregnant and postpartum females using fetuses and type 1 CA from April to November. The fertility rate of the females captured after the second pregnancy season was 90.3 % during the pregnancy period and 100 % during the non-pregnancy period. The high fertility rate of adult females suggests that intensive adult female harvesting is needed. Our new method to determine fertility rates contributes to developing a monitoring system to adequately control Japanese wild boar population.     

Amyloplast displacement is necessary for gravisensing in Arabidopsis shoots as revealed by a centrifuge microscope

The starch‐statolith hypothesis proposes that starch‐filled amyloplasts act as statoliths in plant gravisensing, moving in response to the gravity vector and signaling its direction. However, recent studies suggest that amyloplasts show continuous, complex movements in Arabidopsis shoots, contradicting the idea of a so‐called ‘static’ or ‘settled’ statolith. Here, we show that amyloplast movement underlies shoot gravisensing by using a custom‐designed centrifuge microscope in combination with analysis of gravitropic mutants. The centrifuge microscope revealed that sedimentary movements of amyloplasts under hypergravity conditions are linearly correlated with gravitropic curvature in wild‐type stems. We next analyzed the hypergravity response in the shoot gravitropism 2 (sgr2) mutant, which exhibits neither a shoot gravitropic response nor amyloplast sedimentation at 1  g . sgr2 mutants were able to sense and respond to gravity under 30  g conditions, during which the amyloplasts sedimented. These findings are consistent with amyloplast redistribution resulting from gravity‐driven movements triggering shoot gravisensing. To further support this idea, we examined two additional gravitropic mutants, phosphoglucomutase (pgm) and sgr9, which show abnormal amyloplast distribution and reduced gravitropism at 1  g . We found that the correlation between hypergravity‐induced amyloplast sedimentation and gravitropic curvature of these mutants was identical to that of wild‐type plants. These observations suggest that Arabidopsis shoots have a gravisensing mechanism that linearly converts the number of amyloplasts that settle to the ‘bottom’ of the cell into gravitropic signals. Further, the restoration of the gravitropic response by hypergravity in the gravitropic mutants that we tested indicates that these lines probably have a functional gravisensing mechanism that is not triggered at 1  g .     

Lytic Enzymes toward Aniline-assimilating Rhodococcus erythro-polis AN-13: Purification and Characterization

Three lytic enzymes, C-2, C-4 and C-5, capable of lysing cells of Rhodococcus erythropolis AN-13 were purified from the cultural filtrate of Flavobacterium species SH-548 by (NH₄)₂S0₄ fractionation and column chromatographies on CM-Toyopearl and SP-Sephadex. The three purified enzymes gave single protein bands on polyacrylamide gels. C-4 and C-5 were stable between pH 3.0 and 12.5, and C-2 between pH 5.5 and 11.0. The molecular weights of C-4 and C-5 were 26,000 and that of C-2 was 36,000, as judged on sodium dodecylsulfate-polyacrylamide gel electrophoresis. C-4 and C-5 also showed proteolytic activity toward casein, but C-2 did not exhibit such activity. C-2 showed higher specific lytic activity toward cells of R. erythropolis AN-13 than C-4 and C-5.     

Significance of the Tryptophan Residue at the Low Affinity Saccharide-binding Site of Ricin E

Chemical modification of tryptophan residues in ricin E was investigated with regard to saccharide-binding. Two out of ten tryptophan residues in ricin E were modified with N- bromosuccinimide at pH 4.5 in the absence of specific saccharide accompanied by a marked decrease in the cytoagglutinating activity. Such a loss of the cytoagglutinating activity was found to be principally due to the oxidation of one tryptophan residue per B-chain. In the presence of lactose, one tryptophan residue/mol was protected from the modification with retention of a fairly high cytoagglutinating activity. However, G a IN Ac did not show such a protective effect. The binding of lactose to ricin E altered the environment of the tryptophan residue at the low affinity binding site of ricin E, leading to a blue shift of the fluorescence spectrum and an UV-difference spectrum with a maximum at 290 nm and a trough at 300 nm. The ability to generate such spectroscopic changes induced by lactose was retained in the derivative in which one tryptophan residue/mol was oxidized in the presence of lactose, but not in the derivative in which two tryptophan residues/mol were oxidized in the absence of lactose. Based on these results, it is suggested that one of the two surface-localized tryptophan residues is responsible for saccharide binding at the low affinity binding site of ricin E, which can bind lactose but lacks the ability to bind GalNAc.