Studies on the behavior of meiotic protoplasts

Meiotic protoplasts obtained from lily microsporocytes in late prophase to telophase I were cultured in an enzyme solution which prevents formation of a cell wall around the protoplsts. The removal of the surface wall interfered with nuclear and cell division when the wall was removed prior to metaphase. The main effects were non-segregation of chromosomes and aberrant cytokinesis. In contrast, the absence of a cell wall during the later periods in which actual segregation of the nucleus and cytoplasm takes place did not interfere with the spindle function. The regular process was accomplished through the formation of a cell plate or septum, and 2 hemispheric daughter protoplasts were formed. After that, a furrow was usually formed at the septum in the absence of a surrounding cell wall, and the protoplasts became dumbbell shaped. Some abnormal behavior was also observed using the time lapse technique.     

Group A Streptococcus modulates RAB1- and PIK3C3 complex-dependent autophagy

ABSTRACT

Autophagy selectively targets invading bacteria to defend cells, whereas bacterial pathogens counteract autophagy to survive in cells. The initiation of canonical autophagy involves the PIK3C3 complex, but autophagy targeting Group A Streptococcus (GAS) is PIK3C3-independent. We report that GAS infection elicits both PIK3C3-dependent and -independent autophagy, and that the GAS effector NAD-glycohydrolase (Nga) selectively modulates PIK3C3-dependent autophagy. GAS regulates starvation-induced (canonical) PIK3C3-dependent autophagy by secreting streptolysin O and Nga, and Nga also suppresses PIK3C3-dependent GAS-targeting-autophagosome formation during early infection and facilitates intracellular proliferation. This Nga-sensitive autophagosome formation involves the ATG14-containing PIK3C3 complex and RAB1 GTPase, which are both dispensable for Nga-insensitive RAB9A/RAB17-positive autophagosome formation. Furthermore, although MTOR inhibition and subsequent activation of ULK1, BECN1, and ATG14 occur during GAS infection, ATG14 recruitment to GAS is impaired, suggesting that Nga inhibits the recruitment of ATG14-containing PIK3C3 complexes to autophagosome-formation sites. Our findings reveal not only a previously unrecognized GAS-host interaction that modulates canonical autophagy, but also the existence of multiple autophagy pathways, using distinct regulators, targeting bacterial infection.

Abbreviations: ATG5: autophagy related 5; ATG14: autophagy related 14; ATG16L1: autophagy related 16 like 1; BECN1: beclin 1; CALCOCO2: calcium binding and coiled-coil domain 2; GAS: group A streptococcus; GcAV: GAS-containing autophagosome-like vacuole; LAMP1: lysosomal associated membrane protein 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MTORC1: mechanistic target of rapamycin kinase complex 1; Nga: NAD-glycohydrolase; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; PtdIns3P: phosphatidylinositol-3-phosphate; PtdIns4P: phosphatidylinositol-4-phosphate; RAB: RAB, member RAS oncogene GTPases; RAB1A: RAB1A, member RAS oncogene family; RAB11A: RAB11A, member RAS oncogene family; RAB17: RAB17, member RAS oncogene family; RAB24: RAB24, member RAS oncogene family; RPS6KB1: ribosomal protein S6 kinase B1; SLO: streptolysin O; SQSTM1: sequestosome 1; ULK1: unc-51 like autophagy activating kinase 1; WIPI2: WD repeat domain, phosphoinositide interacting 2     

IAA-induced opening of excisedMimosa pulvinules

Movement ofMimosa pudica L. pulvinules was investigated by using excised ones which were placed on a moist filter paper. The pulvinules excised in the morning opened at the addition of IAA (10⁻⁷ M to 10⁻⁴M) in the dark. The lag period for the onset of the opening was about 15 min. Na-acetate buffer (pH 4) also induced the opening of pulvinules in the dark, and the buffer-induced opening was inhibited by the uncouplers of oxidative phosphorylation. Na-MES and Na-citrate buffers (pH 4) did not induce the opening. Pulvinules taken from closed leaves in the evening were less responsive to IAA than those taken from open leaves in the morning. The pulvinules taken in the evening slightly opened with incandescent light (4000 lux), but those preincubated with IAA (10⁻⁷M and 10⁻⁶M) opened distinctly upon the illumination.