Tetsuko Noguchi

Docosahexaenoic Acid Production and Lipid-Body Formation in Schizochytrium limacinum SR21

Schizochytrium limacinum SR21, a thraustochytrid (Labyrinturomycota), is a heterotrophic marine microorganism. SR21 has attracted recent attention because of the production of docosahexaenoic acid (DHA). We obtained highly concentrated SR21 zoospores and successfully observed synchronous growth. We investigated changes of lipid content and fatty acid composition during the growth. The morphological features of the lipid bodies were also described via fluorescent and electron microscopy. The cells developed quickly after zoospore settlement. Lipid bodies developed in accordance with an increase in lipid content during the 8-h synchronous growth. The total lipid was composed mainly of triacylglycerol, sterol esters, and phosphatidylcholine. The proportion of neutral lipids (triacylglycerol and sterol esters) in the total lipid was fairly constant during growth. The fatty acid composition of neutral lipids, primary components of the lipid body, and phospholipids, primary components of the cell membranes, was nearly unchanged during the synchronous growth. However, the DHA content of the phospholipids decreased drastically after a 10-day culture. Electron micrographs prepared using a high-pressure freeze substitution technique revealed a fine structure of light- and dark-staining bands inside the lipid bodies in many stages of the cells.

Transformation of the Golgi apparatus in the cell cycle, especially at the resting and earliest developmental stages of a green alga,Micrasterias americana

SummaryTransformation of the Golgi apparatus inMicrasterias americana at various stages after full growth and at the earliest stage of cell growth was investigated using an electron microscope. Silver-hexamine staining and the acid phosphatase (ACPase) test were also carried out. In cells cultured for two days after full growth, dictyosomes began to produce hypertrophied vesicles (HVs) along their five peripheral reagions. The HVs contained fibrous material, which was stained by silver-hexamine, and small granules which reacted with ACPase. The HVs were pinched off the dictyosomes and fused with each other and with the vacuoles. In the earliest stage of cell growth, the cisternae of the dictyosomes were stretched in one direction, which modified the shape from circular to elliptical and the dictyosomes curved along the long axis of the ellipse. These curved dictyosomes which produced middle sized vesicles (MVs) from the distal networks, divided into two identical parts along the short axis of the ellipse.

Consumption of lipid granules and formation of vacuoles in the pollen tube ofTradescantia reflexa

SummaryThe ultrastructure in pollen tubes ofTradescantia reflexa was studied with special attention to the consumption of lipid granules and the formation of vacuoles. Electron dense, thin vesicles (EDTV) were developed from the small spherical vesicles. ER-associated lipid granules attached to both surfaces of the EDTV. Then, the EDTV became bottle-shaped, surrounding a part of cytoplasm. In the compartment surrounded by the membranes of EDTV, lipid granules were decomposed and finally disappeared. Protuberances were formed at the terminals of the expanded membranes of the EDTV and at the delimiting membranes of the compartment. These protuberances were pinched off from the membranes to form vesicles. These vesicles were transported from the site of origin to destinations in the pollen grain where they fused with each other to form vacuoles.

DIVISION APPARATUS OF THE CHLOROPLAST IN NANNOCHLORIS BACILLARIS (CHLOROPHYTA)1

Chloroplast division in Nannochloris bacillaris Naumann (Chlorophyta) was examined by electron microscopy after preparation of samples by freeze‐substitution. A pair of belts appeared on the surface of the outer and inner envelope membranes at the middle of the chloroplast. These belts seemed to be constructed of thin fibrils that run parallel to the longitudinal direction of the belts. The outer fibrillar belt increased in width as the constriction of the chloroplast advanced. It appears that the fibrillar belt is the division apparatus of the chloroplast. It encircles the chloroplast and finally divides the chloroplast in two as the diameter of the belt decreases.

Transformation of Lipid Bodies Related to Hydrocarbon Accumulation in a Green Alga, Botryococcus braunii (Race B)

The colonial microalga Botryococcus braunii accumulates large quantities of hydrocarbons mainly in the extracellular space; most other oleaginous microalgae store lipids in the cytoplasm. Botryococcus braunii is classified into three principal races (A, B, and L) based on the types of hydrocarbons. Race B has attracted the most attention as an alternative to petroleum by its higher hydrocarbon contents than the other races and its hydrocarbon components, botryococcenes and methylsqualenes, both can be readily converted into biofuels. We studied race B using fluorescence and electron microscopy, and clarify the stage when extracellular hydrocarbon accumulation occurs during the cell cycle, in a correlation with the behavior and structural changes of the lipid bodies and discussed development of the algal colony. New accumulation of lipids on the cell surface occurred after cell division in the basolateral region of daughter cells. While lipid bodies were observed throughout the cell cycle, their size and inclusions were dynamically changing. When cells began dividing, the lipid bodies increased in size and inclusions until the extracellular accumulation of lipids started. Most of the lipids disappeared from the cytoplasm concomitant with the extracellular accumulation, and then reformed. We therefore hypothesize that lipid bodies produced during the growth of B. braunii are related to lipid secretion. New lipids secreted at the cell surface formed layers of oil droplets, to a maximum depth of six layers, and fused to form flattened, continuous sheets. The sheets that combined a pair of daughter cells remained during successive cellular divisions and the colony increased in size with increasing number of cells.

Active Hydrocarbon Biosynthesis and Accumulation in a Green Alga, Botryococcus braunii (Race A)

ABSTRACT Among oleaginous microalgae, the colonial green alga Botryococcus braunii accumulates especially large quantities of hydrocarbons. This accumulation may be achieved more by storage of lipids in the extracellular space rather than in the cytoplasm, as is the case for all other examined oleaginous microalgae. The stage of hydrocarbon synthesis during the cell cycle was determined by autoradiography. The cell cycle of B. braunii race A was synchronized by aminouracil treatment, and cells were taken at various stages in the cell cycle and cultured in a medium containing [14C]acetate. Incorporation of 14C into hydrocarbons was detected. The highest labeling occurred just after septum formation, when it was about 2.6 times the rate during interphase. Fluorescent and electron microscopy revealed that new lipid accumulation on the cell surface occurred during at least two different growth stages and sites of cells. Lipid bodies in the cytoplasm were not prominent in interphase cells. These lipid bodies then increased in number, size, and inclusions, reaching maximum values just before the first lipid accumulation on the cell surface at the cell apex. Most of them disappeared from the cytoplasm concomitant with the second new accumulation at the basolateral region, where extracellular lipids continuously accumulated. The rough endoplasmic reticulum near the plasma membrane is prominent in B. braunii, and the endoplasmic reticulum was often in contact with both a chloroplast and lipid bodies in cells with increasing numbers of lipid bodies. We discuss the transport pathway of precursors of extracellular hydrocarbons in race A.

Effects of brefeldin A on the Golgi apparatus, the nuclear envelope, and the endoplasmic reticulum in a green alga,Scenedesmus acutus

SummaryThe effects of brefeldin A (BFA) on the structure of the Golgi apparatus, the nuclear envelope, and the endoplasmic reticulum (ER), and on the thiamine pyrophosphatase (TPPase) activity in these organelles were examined in a green alga,Scenedesmus acutus, to obtain evidence for the existence of a retrograde transport from the Golgi apparatus to the ER via the nuclear envelope. InScenedesmus, Golgi bodies are situated close to the nuclear envelope throughout the cell cycle and receive the transition vesicles not directly from the ER, but from the nuclear envelope. BFA induced the disassembly of Golgi bodies and an increase in the ER cisternae at the trans-side of decomposed Golgi bodies in interphase cells and multinuclear cells before septum formation. The accumulated ER cisternae connected to the nuclear envelope at one part. TPPase activity was detected in all cisternae of Golgi bodies, but not in the nuclear envelope or the ER in nontreated cells. On the contrary, in BFA-treated cells, TPPase activity was detected in the nuclear envelope and the ER in addition to the decomposed Golgi bodies. When septum-forming cells were treated with BFA, the disassembly of Golgi bodies was less than that in interphase cells, and TPPase activity was detected in the Golgi cisternae but not in the nuclear envelope or the ER. These results suggest mat BFA blocks the anterograde transport from the nuclear envelope to the Golgi bodies but does not block the retrograde transport from the Golgi bodies to the nuclear envelope in interphase and multinuclear cells.

Cortical microtubules and cortical microfilaments in the green alga,Micrasterias pinnatifida

SummaryCortical microtubules and cortical microfilaments were visualized in cells ofMicrasterias pinnatifida treated by freeze-substitution, and the pattern of their distribution was reconstructed from serial sections. Most cortical microtubules accompanied the long microfilaments that ran parallel to the microtubules. Cortical microfilaments not accompanied by the microtubules were also found. They were short and slightly curved. Both types of cortical microfilament were not grouped into bundles, and were 6–7 nm in diameter, a value that corresponds to the diameter of filaments of F-actin.

Transformation of trans-Golgi Network During the Cell Cycle in a Green Alga, Botryococcus braunii

trans-Golgi network (TGN), and the changes in its structure and behavior throughout the cell cycle of a unicellular green alga, Botryococcus braunii, were examined with deep-etching replicas and in cryo-fixed/freeze-substituted specimens. In interphase cells, the TGN consisted of a hemispherically shaped cisterna (TGN-cisterna) with regularly distributed pores on the surface and a tubular network (TGN-tubules) with clathrin-coated vesicles. The TGNs changed their structure drastically throughout the cell cycle. The TGN-cisterna disappeared from the beginning of nuclear division to the completion of the cell wall, in contrast that TGN-tubules with the clathrin-coated vesicles were always observed. The TGN-tubules produced at least five other kinds of vesicles depending on the stage of the cell cycle: 200-nm vesicles with fibrillar substances and multivesicular bodies in interphase, 180–240 nm vesicles during cell division, and 400–450 nm vesicles containing fibrils and small masses of electron-dense substances, and 200-nm vesicles containing electron-dense spherical substances just after cell division. During cell wall formation, TGN-tubules were small and had only a few clathrin-coated vesicles. After cell wall formation, TGN-tubules grew and a TGN-cisterna with pores appeared again.

Formation and decomposition of vacuoles inBotryococcus in relation to the trans-Golgi network

SummaryThe formation and the decomposition of vacuoles in a member of Xanthophyceae,Botryococcus braunii, were examined by light and electron microscopy. Particles around the nucleus were identified as vacuoles from their stainability with neutral red. These particles disappeared during cell division. They reacted positively in an activity test for acid phosphatase. Electron microscopy revealed the presence of spherical vacuoles around the nucleus. During cell division, these vacuoles seemed to be decomposed by the ER which surrounded the vacuoles. Soon after this decomposition, many immature multivesicular bodies (MVBs) appeared to develop from the trans-Golgi network (TGN) and were pinched off from the TGN. These immature MVBs took up small vesicles in them as they grew into the mature MVBs. Mature MVBs took up and digested the surrounding cytoplasm, fused with one another, and eventually became the vacuoles.