Biosynthetic and metabolic activities of some organelles in Papaver somniferum latex

The biosynthesis of the five major alkaloids of the opium poppy (Papaver somniferum L. from radioactive dihydroxyphenylalanine has been studied in the 1000 g, 10 000 g, 100 000 g pellets and the 100 000 g supernatant fractions of the capsule latex. A normal poppy variety as well as one which produces only traces of alkaloids were used. Definite evidence of biosynthesis was obtained for both varieties but only in the 1 000 g pellet (as previously reported . None was found in the other fractions although electron microscopy showed that organelles, including vesicles, were present. The amounts of alkaloid biosynthesized however were very small relative to the amounts involved in the rapid changes already reported for the developing capsules. In contrast, all fractions of the latex were able to metabolize T-morphine in vitro, with the 100 000 g supernatant showing the highest activity and the amounts involved were also consistent with the changes found in the living plant.     

Cilia and the life of ctenophores

Ctenophores, or comb jellies, are a distinct phylum of marine zooplankton with eight meridional rows of giant locomotory comb plates. Comb plates are the largest ciliary structures known, and provide unique experimental advantages for investigating the biology of cilia. Here, I review published and unpublished work on how ctenophores exploit both motile and sensory functions of cilia for much of their behavior. The long‐standing problem of ciliary coordination has been elucidated by experiments on a variety of ctenophores. The statocyst of ctenophores is an example of how mechanosensory properties of motile cilia orient animals to the direction of gravity. Excitation or inhibition of comb row beating provides adaptive locomotory responses, and global reversal of beat direction causes escape swimming. The diverse types of prey and feeding mechanisms of ctenophores are related to radiation in body form and morphology. The cydippid Pleurobrachia catches copepods on tentacles and undergoes unilateral ciliary reversal to sweep prey into its mouth. Mnemiopsis uses broad muscular lobes and ciliated auricles to capture and ingest prey. Beroë has giant smooth muscles and toothed macrocilia to rapidly engulf or bite through ctenophore prey, and uses reversible tissue adhesion to keep its mouth closed while swimming. Ciliary motor responses are calcium‐dependent, triggered by voltage‐activated calcium channels located along the length (reversed beating) or at the base (activation of beating) of ciliary membranes. Ciliary and muscular responses to stimuli are regulated by epithelial and mesogleal nerve nets with ultrastructurally identifiable synapses onto effector cells. Post‐embryonic patterns of comb row development in larval and adult stages are described and compared with regeneration of comb plates after surgical removal. Truly, cilia and ctenophores, like love and marriage, go together like a horse and carriage.     

Cellular organelles facilitate dimerization of a newly identified Arf from Chlamydomonas reinhardtii

GTPases of the Ras superfamily regulate a wide variety of cellular processes including vesicular transport and various secretory pathways of the cell. ADP – ribosylation factor (ARF) belongs to one of the five major families of the Ras superfamily and serves as an important component of vesicle formation and transport machinery of the cells. The binding of GTP to these Arfs and its subsequent hydrolysis, induces conformational changes in these proteins leading to their enzymatic activities. The dimeric form of Arf is associated with membrane pinch‐off during vesicle formation. In this report, we have identified an arf gene from the unicellular green alga Chlamydomonas reinhardtii, CrArf, and showed that the oligomeric state of the protein in C. renhardtii is modulated by the cellular membrane environment of the organism. Protein cross‐linking experiments showed that the purified recombinant CrArf has the ability to form a dimer. Both the 20‐kDa monomeric and 40‐kDa dimeric forms of CrArf were recognized from Chlamydomonas total cell lysate (CrTLC) and purified recombinant CrArf by the CrArf specific antibody. The membranous environment of the cell appeared to facilitate dimerization of the CrArf, as dimeric form was found exclusively associated with the membrane bound organelles. The subcellular localization studies in Chlamydomonas suggested that CrArf mainly localized in the cytosol and was mislocalized in vesicle transport machinery inhibitor treated cells. This research sheds light on the importance of the cellular membrane environment for regulating the oligomeric state of CrArf protein in this organism and associated functional role.     

MINIPODIA AND ROSETTE CONTACTS ARE ADHESIVE ORGANELLES PRESENT IN FREE-LIVING AMOEBAE

Using scanning electron microscopy, Amoeba proteus cells migrating on the glass have been shown to develop dense coats of minipodia, which are discrete microprotrusions up to 8 microm long and approximately 0.5 microm across. They cover the middle-anterior area of the ventral cell surface, i.e. the region previously determined as the zone of most efficient adhesion of an amoeba to its substratum. Minipodia are sparse underneath the frontal zone and lacking from the tail region. In amoebae that adhere to the glass without moving, have just started moving, or show unstable motor polarity, minipodia are grouped in rosette contacts, cauliflower-like papillae composed of supporting platforms with crowns of minipodia emerging from them. Both structures abound with cytoskeletal F-actin, as shown by staining with fluorescein-conjugated phalloidin. Amoebae experimentally prevented from adhering to the substratum neither develop discrete minipodia nor rosette contacts.     

Involvement of Vps33a in the Fusion of Uroplakin-Degrading Multivesicular Bodies with Lysosomes

The apical surface of the terminally differentiated mouse bladder urothelium is largely covered by urothelial plaques, consisting of hexagonally packed 16-nm uroplakin particles. These plaques are delivered to the cell surface by fusiform vesicles (FVs) that are the most abundant cytoplasmic organelles. We have analyzed the functional involvement of several proteins in the apical delivery and endocytic degradation of uroplakin proteins. Although FVs have an acidified lumen and Rab27b, which localizes to these organelles, is known to be involved in the targeting of lysosome-related organelles (LROs), FVs are CD63 negative and are therefore not typical LROs. Vps33a is a Sec1-related protein that plays a role in vesicular transport to the lysosomal compartment. A point mutation in mouse Vps33a (Buff mouse) causes albinism and bleeding (Hermansky-Pudlak syndrome) because of abnormalities in the trafficking of melanosomes and platelets. These Buff mice showed a novel phenotype observed in urothelial umbrella cells, where the uroplakin-delivering FVs were almost completely replaced by Rab27b-negative multivesicular bodies (MVBs) involved in uroplakin degradation. MVB accumulation leads to an increase in the amounts of uroplakins, Lysosomal-associated membrane protein (LAMP)-1/2, and the activities of β-hexosaminidase and β-glucocerebrosidase. These results suggest that FVs can be regarded as specialized secretory granules that deliver crystalline arrays of uroplakins to the cell surface, and that the Vps33a mutation interferes with the fusion of MVBs with mature lysosomes thus blocking uroplakin degradation.     

Comparison of active transport in neuronal axons and dendrites

This paper presents a theoretical study, based on modified Smith-Simmons equations, that compares transport of intracellular organelles in two different neurite outgrowths, dendrites and axons. It is demonstrated that the difference in microtubule polarity orientations in dendrites and axons has significant implications on motor-assisted transport in these neurite outgrowths. The developed approach presents a qualitative theoretical basis for understanding important questions such as why axons exhibit almost an unlimited grows potential in vitro while dendrites remain relatively short. It is shown that the difference in a microtubule polarity arrangement between axons and dendrites may be a regulatory mechanism for limiting dendritic growth. Other biological implications of the developed theory as well as other possible reasons for the difference in microtubule structure between axons and dendrites are discussed.     

Mitochondrial distribution and microtubule organization in fertilized and cloned porcine embryos: implications for developmental potential

Mitochondrial distribution and microtubule organization were examined in porcine oocytes after parthenogenesis, fertilization and somatic cell nuclear transfer (SCNT). Our results revealed that mitochondria are translocated from the oocyte's cortex to the perinuclear area by microtubules that either constitute the sperm aster in in vitro-fertilized (IVF) oocytes or originate from the donor cell centrosomes in SCNT oocytes. The ability to translocate mitochondria to the perinuclear area was lower in SCNT oocytes than in IVF oocytes. Sperm-induced activation rather than electrical activation of SCNT oocytes as well as the presence of the oocyte spindle enhanced perinuclear mitochondrial association with reconstructed nuclei, while removal of the oocyte spindle prior to sperm penetration decreased mitochondrial association with male pronuclei without having an apparent effect on microtubules. We conclude that factors derived from spermatozoa and oocyte spindles may affect the ability of zygotic microtubules to translocate mitochondria after IVF and SCNT in porcine oocytes. Mitochondrial association with pronuclei was positively related with embryo development after IVF. The reduced mitochondrial association with nuclei in SCNT oocytes may be one of the reasons for the low cloning efficiency which could be corrected by adding yet to be identified, sperm-derived factors that are normally present during physiological fertilization.     

Acridine orange accumulation in acid organelles of normal and vacuolated frog skeletal muscle fibres

The spatial distribution of acid membrane organelles and their relationships with normal and vacuolated transverse tubules has been studied in living frog skeletal muscle fibres using confocal microscopy. Acridine orange (AO) was used to evaluate acid compartments, while a lipophilic styryl dye, RH 414, was employed to stain the membranes of the T-system. AO accumulated in numerous spherical granules located near the poles of nuclei and between myofibrils where they were arranged in short parallel rows, triplets or pairs. AO granules could be divided into three groups: green (monomeric AO), red (aggregated AO), and mixed green/red. As demonstrated by lambda-scanning, most granules were mixed. Double staining of muscle fibres with AO and RH 414 revealed almost all AO granules located near the transverse tubules. Vacuolation of the T-system was induced by glycerol loading and subsequent removal. The close juxtaposition of AO granules and the T-system was preserved in vacuolated fibres. The lumens of vacuoles did not accumulate AO. It is concluded that AO granules represent an accumulation of AO in lysosome-related organelles and fragmented Golgi apparatus and a possible functional role of the spatial distribution of such acidic compartments is discussed.     

Mitochondria-derived organelles in the diplomonad fish parasite Spironucleus vortens

In some eukaryotes, mitochondria have become modified during evolution to yield derived organelles (MDOs) of a similar size (hydrogenosomes), or extremely reduced to produce tiny cellular vesicles (mitosomes). The current study provides evidence for the presence of MDOs in the highly infectious fish pathogen Spironucleus vortens, an organism that produces H₂ and is shown here to have no detectable cytochromes. Transmission electron microscopy (TEM) reveals that S. vortens trophozoites contain electron-dense, membranous structures sometimes with an electron-dense core (200 nm–1 μm), resembling the hydrogenosomes previously described in other protists from habitats deficient in O₂. Confocal microscopy establishes that these organelles exhibit autofluorescence emission spectra similar to flavoprotein constituents previously described for mitochondria and also present in hydrogenosomes. These organelles possess a membrane potential and are labelled by a fluorescently labeled antibody against Fe-hydrogenase from Blastocystis hominis. Heterologous antibodies raised to mitochondrial proteins frataxin and Isu1, also exhibit a discrete punctate pattern of localization in S. vortens; however these labelled structures are distinctly smaller (90–150 nm) than hydrogenosomes as observed previously in other organisms. TEM confirms the presence of double-membrane bounded organelles of this smaller size. In addition, strong background immunostaining occurs in the cytosol for frataxin and Isu1, and labelling by anti-ferredoxin antibody is generally distributed and not specifically localized except for at the anterior polar region. This suggests that some of the functions traditionally attributed to such MDOs may also occur elsewhere. The specialized parasitic life-style of S. vortens may necessitate more complex intracellular compartmentation of redox reactions than previously recognized. Control of infection requires biochemical characterization of redox-related organelles.     

The initiative on Model Organism Proteomes (iMOP) Session September 6, 2011, Geneva, Switzerland

iMOP--the Initiative on Model Organism Proteomes--was accepted as a new HUPO initiative at the Ninth HUPO meeting in Sydney in 2010. A goal of iMOP is to integrate research groups working on a great diversity of species into a model organism community. At the Tenth HUPO meeting in Geneva this variety was reflected in the iMOP session on Tuesday September 6, 2011. The presentations covered the quantitative proteome database PaxDb, proteomics projects studying farm animals, Arabidopsis thaliana, as well as host-pathogen interactions.