Correlated waves of actin filaments and PIP3 in Dictyostelium cells

Chemotaxis-deficient amiB-null mutant Dictyostelium cells show two distinct movements: (1) they extend protrusions randomly without net displacements; (2) they migrate persistently and unidirectionally in a keratocyte-like manner. Here, we monitored the intracellular distribution of phosphatidylinositol (3,4,5)-trisphosphate (PIP(3)) to gain insight into roles PIP(3) plays in those spontaneous motilities. In keratocyte-like cells, PIP(3) showed convex distribution over the basal membrane, with no anterior enrichment. In stalled cells, as well as in wild type cells, PIP(3) repeated wave-like changes, including emergence, expansion and disappearance, on the basal membrane. The waves induced lamellipodia when they approached the cell edge, and the advancing speed of the waves was comparable to the migration speed of the keratocyte-like cells. LY294002, an inhibitor of PI3 kinase, abolished PIP(3) waves in stalled cells and stopped keratocyte-like cells. These results together suggested that keratocyte-like cells are "surfing" on the PIP(3) waves by coupling steady lamellipodial protrusions to the PIP(3) waves. Simultaneous live observation of actin filaments and PIP(3) in wild type or stalled amiB(-) cells indicated that the PIP(3) waves were correlated with wave-like distributions of actin filaments. Most notably, PIP(3) waves often followed actin waves, suggesting that PIP(3) induces local depolymerization of actin filaments. Consistent with this idea, cortical accumulation of PIP(3) was often correlated with local retraction of the periphery. We propose that the waves of PIP(3) and actin filaments are loosely coupled with each other and play important roles in generating spontaneous cell polarity.     

Characterization of Nontoxic-Nonhemagglutinin Component of the Two Types of Progenitor Toxin (M and L) Produced by Clostridium botulinum Type D CB-16

A 9.8-kbp DNA fragment which contained a neurotoxin gene and its upstream region was cloned from Clostridium botulinum type D strain CB-16. Nucleotide sequencing of the fragment revealed that genes encoding for hemagglutinin (HA) subcomponents and one for a nontoxic-nonhemagglutinin (NTNH) component were located upstream of the neurotoxin gene. This strain produced two toxins of different molecular size (approximately 300 kDa and 500 kDa) which were designated as progenitor toxins (M and L toxins). The molecular size of the NTNH component of L toxin was approximately 130 kDa on SDS-PAGE and its N-terminal amino acid sequence was M-D-I-N-D-D-L-N-I-N-S-P-V-D-N-K-N-V-V-I which agreed with that deduced from the nucleotide sequence. In contrast, the M toxin had a 115-kDa NTNH component whose N-terminal sequence was S-T-I-P-F-P-F-G-G-Y-R-E-T-N-Y-I-E, corresponding to the sequence from Ser141 of the deduced sequence. A 15-kDa fragment, which was found to be associated with an M toxin preparation, possessed the same N-terminal amino acid sequence as that of the 130-kDa NTNH component. Furthermore, five major fragments generated by limited proteolysis with V8 protease were shown to have N-terminal amino acid sequences identical to those deduced from the nucleotide sequence of 130-kDa NTNH. These results indicate that the 130-kDa NTNH of the L toxin is cleaved at a unique site, between Thr and Ser, leading to the 115-kDa NTNH of the M toxin.     

Signal strength controls the rate of polarization within CTLs during killing

Cytotoxic T lymphocytes (CTLs) are key effector cells in the immune response against viruses and cancers, killing targets with high precision. Target cell recognition by CTL triggers rapid polarization of intracellular organelles toward the synapse formed with the target cell, delivering cytolytic granules to the immune synapse. Single amino acid changes within peptides binding MHC class I (pMHCs) are sufficient to modulate the degree of killing, but exactly how this impacts the choreography of centrosome polarization and granule delivery to the target cell remains poorly characterized. Here we use 4D imaging and find that the pathways orchestrating killing within CTL are conserved irrespective of the signal strength. However, the rate of initiation along these pathways varies with signal strength. We find that increased strength of signal leads to an increased proportion of CTLs with prolonged dwell times, initial Ca²⁺ fluxes, centrosome docking, and granule polarization. Hence, TCR signal strength modulates the rate but not organization of effector CTL responses.     

Factor Structure of the Japanese Version of the Edinburgh Postnatal Depression Scale in the Postpartum Period

Background

The Edinburgh Postnatal Depression Scale (EPDS) is a widely used screening tool for postpartum depression (PPD). Although the reliability and validity of EPDS in Japanese has been confirmed and the prevalence of PPD is found to be about the same as Western countries, the factor structure of the Japanese version of EPDS has not been elucidated yet.

Methods

690 Japanese mothers completed all items of the EPDS at 1 month postpartum. We divided them randomly into two sample sets. The first sample set (n = 345) was used for exploratory factor analysis, and the second sample set was used (n = 345) for confirmatory factor analysis.

Results

The result of exploratory factor analysis indicated a three-factor model consisting of anxiety, depression and anhedonia. The results of confirmatory factor analysis suggested that the anxiety and anhedonia factors existed for EPDS in a sample of Japanese women at 1 month postpartum. The depression factor varies by the models of acceptable fit.

Conclusions

We examined EPDS scores. As a result, “anxiety” and “anhedonia” exist for EPDS among postpartum women in Japan as already reported in Western countries. Cross-cultural research is needed for future research.     

Comparison of Attenuation of Striated Muscle between Postmortem and Antemortem Computed Tomography: Results of a Longitudinal Study

Objective

We evaluated the postmortem changes of striated muscle by comparing computed tomography (CT) images obtained postmortem and antemortem in the same patients.

Materials and Methods

We studied 33 consecutive patients who underwent antemortem CT, postmortem CT, and pathological autopsy in our tertiary care hospital between April 2009 and December 2010. Postmortem CT was performed within 20 h after death and was followed by pathological autopsy. Pathological autopsy confirmed the absence of muscular diseases such as amyotrophic lateral sclerosis, muscular dystrophy, myositis, and myasthenia, in all of the patients. The CT attenuation values of four cardiac muscle sites (anterior wall of the left ventricle, left ventricular free wall, posterior wall of the left ventricle, and the ventricular septum) and two skeletal muscle sites (the pectoralis major muscle and the erector spinae muscle) were compared between antemortem and postmortem CT using paired t test.

Results

Striated muscle had significantly greater attenuation on postmortem CT than on antemortem CT (P<0.001) in all six tissue sites. No significant association was found between postmortem change in the CT attenuation of striated muscle and gender, age, or elapsed time since death.

Conclusion

This is the first longitudinal study to show hyperattenuation of striated muscle on postmortem CT images compared with antemortem CT images in the same patients.     

Cyclin B-cdk1 controls pronuclear union in interphase

In sexual reproduction, the union of the male and female pronuclei occurs in fertilized eggs to mix genetic materials derived from both parents, thereby creating a new genome for the next generation [1-4]. The process leading to pronuclear union consists of pronuclear congression, which depends on astral microtubules derived from sperm centrosome [5-8], and the subsequent pronuclear fusion or karyogamy. The union process progresses in parallel with the first embryonic cell cycle, but the molecular mechanisms involved are poorly understood. Here, we devise a labeling method with Dendra2 to track both pronuclei individually in living starfish eggs. Although pronuclear union naturally proceeds while G1 arrest is released by fertilization and S phase progresses [9], we show that the cell-cycle resumption and progression are not prerequisites for pronuclear union. However, low levels of cyclin B- (but not cyclin A-) Cdk1 activity are detectable even in interphase, and are indispensable for pronuclear union, by contributing at least to pronuclear congression through formation of sperm aster. Pronuclear congression thus requires the activity of M-phase cell-cycle regulator in interphase, independently of the cell-cycle regulation. These findings not only provide a clue to the regulatory aspect of creation of new genome with fertilization, but also reveal a novel role for the M-phase Cdk1 during interphase.