Lipotubuloids – Structure and Function

In the 19th and 20th centuries these structures were called „elaioplasts” according to Wakker (1888) who introduced this term with regard to nucleus-size lipid bodies present in Vanilla planifolia leaf epidermis which strongly refracted light. Since they are clearly visible under light microscope (Fig. 1A,B) they became the focus of interest in those days and were described in about 120 monoand dicotyledonous plant species. Also in our laboratory “elaioplasts” were for the first time observed in 12 Gentiana species (Kwiatkowska, 1959, 1961) and in Dahlia variabilis (Kwiatkowska, 1963).


Introduction
In the 19th and 20th centuries these structures were called "elaioplasts" according to Wakker (1888) who introduced this term with regard to nucleus-size lipid bodies present in Vanilla planifolia leaf epidermis which strongly refracted light.Since they are clearly visible under light microscope (Fig. 1A,B) they became the focus of interest in those days and were described in about 120 mono-and dicotyledonous plant species.Also in our laboratory "elaioplasts" were for the first time observed in 12 Gentiana species (Kwiatkowska, 1959(Kwiatkowska, , 1961) ) and in Dahlia variabilis (Kwiatkowska, 1963).Fig. 1.Ornithogalum umbellatum "elaioplast" (lipotubuloid); A -in a living cell; B -after OsO 4 fixation; C -a scheme of epidermis cell with lipotubuloid; G -Golgi apparatus, l -lipotubuloid, lb -lipid bodies, m -mitochondrion, n -nucleus, no -nucleolus, p -plastid, t -tonoplast, v -vacuole; bars: 10 μm.
Elaioplasts are mainly filled with aggregates of osmiophilic granules identified as lipid bodies (Kwiatkowska, 1973a) and surrounded by a phospholipid half unit membrane characteristic only of these structures (Yatsu & Jacks 1972).Differences in the structure and thickness of lipid mono-and bilayer were revealed only after fixation of O. umbellatum epidermis in freshly prepared mixture of OsO 4 and glutaraldehyde (Kwiatkowska, 1973a).
Lipid bodies of plants are covered with proteins, oleosins, which prevent their merging during oil seed germination (Huang, 1996;Hsieh & Huang, 2004).It was shown that the amount of oleosins in a cell determined the size of lipid bodies (Siloto et al., 2006).
Beside lipid bodies and microtubules in this domain there are numerous ribosomes, ER cisternae and vesicles as well as few mitochondria, microbodies and Golgi structures and also autolytic vacuoles later during development.
All the above mentioned lipotubuloids contain the same organelle (Fig. 4) which implies that their presence there is not accidental but results from a functional relationship between them and lipid bodies.The main difference consists in stability of microtubules, those in O. umbellatum are markedly more stable, probably due to the polysaccharide layer covering them, than the others (Kwiatkowska, 1973b).(Kwiatkowska et al., 2010).
In the case of H. albiflos lipotubuloids become visible only after preincubation in 15 μm taxol with microtubule stabilizing buffer (100 mM PIPES, 1 mM MgCl 2 , 5 mM EGTA) pH 7.2 for 2 h prior to fixation in the freshly prepared mixture of 2.5% glutaraldehyde and 1% OsO 4 , followed by postfixation in 1% OsO 4 (Fig. 5).The presence of tubulin around lipid bodies was confirmed by the immunogold method with the anti-tubulin antibody (Kwiatkowska et al., 2010).
In the case of V. planifolia, F. Sieboldiana and A. rosea lipotubuloids were fixed according to the same procedure but without taxol preincubation.anti-tubulin antibody application also gave positive results: gold grains were localized around lipid bodies of lipotubuloids in these plants.
Contrary to O. umbellatum lipotubuloids, microtubules in the other species were mostly observed in cross-sections near lipid bodies, no microtubules between lipid bodies, characteristic of O. umbellatum, were noted.This might be the result of poorer stability of these microtubules as varying stability of microtubules even in the same cell (Kwiatkowska et al., 2011c) is a well known phenomenon resulting, among others, from post-translational tubulin modifications (PTM) such as acetylation, tyrosination, polyglutamylation, polyglycylation, phosphorylation, palmitoylation (Verhey & Gaertig, 2007;Hammond et al., 2008;Fukushima et al., 2009).Nevertheless, we think that it is fully justified to call the structures present in H. albiflos, V. planifolia, F. Sieboldiana A. rosea lipotubuloids.It is highly probable that among "elaioplasts" which are not plastids described in other species further lipotubuloids will be identified.Due to that old papers concerning "elaioplasts" are listed in literature and a table presenting families in which "elaioplasts" were described is included (Table .1) The main subject of our work have been lipid bodies and microtubules in O. umbellatum lipotubuloids which are most stable during fixation for EM.We returned to them after 30 years (during which we concentrated on Chara spermiogenesis) due to the development of new methods and new ideas concerning lipid bodies.

Lipid bodies -Biogenesis and lipid synthesis
Lipid bodies (lipid droplets, oil bodies, spherosomes) are organelles commonly present in plants, animals and some Prokaryote.For many years they were treated only as the source o f e n e r g y , h o w e v e r d u r i n g t h e l a s t d e c a d e t h e y w e r e s h o w n t o p l a y a r o l e i n l i p i d homeostasis and to contain numerous proteins involved in signal transductions as well as proteins transiently stored or degraded (Martin & Parton, 2005;Cermelli et al., 2006;Welte, 2007;Fujimoto et al., 2008).Moreover, they became the focus of interest because disturbances in their functioning cause many serious dieses in humans and animals such as obesity, diabetes, atherosclerosis, cardiovascular disease, allergic inflammation, arthritis, mycobacterial infections, bacterial sepsis, acute respiratory distress syndrome (Bozza et al., 2009).Lipid bodies are also important for plant productivity and biotechnology (Baud & Lepiniec, 2010;Lu et al., 2011).
Cytological studies with the use of light and electron microscopy combined with autoradiography and immunocytochemistry might significantly elucidate lipid bodies biogenesis and function as well as their interactions with other cell organelles.
Lipotubuloids which contain an enormous number of lipid bodies and microtubules are especially suitable experimental material.
Biogenesis of lipid bodies is one of cytological controversies.Some scientists believe that they are formed in cytoplasm as naked droplets (Lung & Weselake, 2006) which are later surrounded by a half unit membrane, others suggest that both ER membranes take part in their biogenesis (Robenek et al., 2011).However, the most common hypothesis is based on old ultrastructural observations (Frey-Wyssling et al., 1963) of plant cells which indicate that ER contributes to lipid bodies formation resulting from the accumulation of lipids between leaflets of a phospholipid bilayer (Fujimoto et al., 2008;Ducharme & Bickel, 2008;Guo et al., 2009;Ohsaki et al., 2009).
In O. umbellatum lipotubuloids lipid bodies are formed in the latter way.This process can be observed in the lipotubuloids enlarging due to de novo formation of lipid bodies during intensive ovary epidermis growth (Kwiatkowska, 1971b).Ovary epidermis cells are characterized by intensive growth without mitotic divisions leading to their 30-fold (by 3000%) enlargement (Fig. 6).It is also closely correlated with lipotubuloid growth (correlation coefficient 0.98) which indicates the important role played by these structures in cells (Kwiatkowska et al., in prep.).
Fig. 6.O. umbellatum ovary epidermis and lipotubuloid development (Kwiatkowska, 1971b) Forming lipid bodies first appear as a gap (Fig. 7A) then change into a lens-like structure grey in color (Fig. 7B,C) .Later it enlarges and becomes greatly osmiophilic, due to that the phospholipid half unit membrane which surrounds it is poorly visible as a thinner and lighter structure than a bilayer membrane (Fig. 7E-G).It appears very clearly later on around mature spherical lipid bodies which are filled with less osmiophilic and more homogenic substance (Fig. 7I,J) as well as in structures which are transient forms between forming and mature lipid bodies (Fig. 7H); these transient forms are connected with ER.However, mature lipid bodies are not linked with ER directly, but through microtubules which are adjacent to ER at one end and to lipid bodies at the other.
The immunogold technique revealed single or aggregated gold grains indicating the presence of diacylglicerol acyltransferase 1 and 2 (DGAT1 and DGAT2) in the specific rough ER regions and which means that these enzymes are synthesized in ER bound ribosomes (Fig. 8) (Kwiatkowska et al., 2011b).More gold grains indicating DGAT1 and DGAT2 were observed in characteristic swollen regions surrounded by thinner membranes at the ends or sides of ER cisternae (Fig. 9).
These pictures seem to present lipid bodies in statu nascendi.They suggest that during their formation enzymes are transported from ER to the surface of lipid bodies.These pictures correspond to gold grains localization indicating DGAT1 and DGAT2 situated at the outside zone of mature lipid bodies (Fig. 10).Thus it seems possible that the last stage of lipid synthesis, transformation of DAG into TAG, can take place in mature lipid bodies similarly as in adipocytes and COS7 fibroblast (Kuerschner et al., 2008).umbellatum lipotubuloids autoradiographic studies with 3 H-palmitic acid at the ultrastructural level (Kwiatkowska et al., 2011b) directly prove that lipid synthesis does take place at the outside of mature lipid bodies (Fig. 11B-J).Silver grains and gold grains resulting from autoradiographic and immunogold reactions, respectively are co-localized at the surface of mature lipid bodies at the half unit membrane/core border which clearly indicate their contribution to lipid synthesis contrary to lipid bodies in statu nascendi in which lipid synthesis is connected directly with ER (see above).
Recent studies with immunogold reaction and 20 nm colloidal gold coupled with anti-lipase antibodies have shown the presence of lipase near lipid bodies surfaces (Fig. 12) (Kwiatkowska et al., in prep.).Similar lipase localization in Ricinus communis was observed by Eastmond (2004) with the use of immunogold method.Lipase is probably responsible for the disappearance of selective labeling of lipotubuloids incubated for 2 h in 3 H-palmitic acid and postincubated for 6 h in the non-radioactive medium (Kwiatkowska, 2004).The mature lipid bodies are approximately of the same size (0.1-0.4 μm) during ovary development in spite of active lipid synthesis due to the dynamic balance between lipid synthesis and lipolysis, however the number of lipid bodies in lipotubuloids which grow significantly, increases (Kwiatkowska et al., 2007).Thus the fact that lipid bodies do not enlarge cannot be treated as an unequivocal proof that lipid synthesis does not occur in them.

Microtubules and lipotubuloid movement
Microtubules as cytoskeleton elements are mostly involved in movement.Lipotubuloids are characterized with very specific and dynamic movement (Kwiatkowska, 1972a).It consists of sometimes very dynamic rotation with varying speed, direction and axis as well as progressive movement (Fig. 13).Lipotubuloid progressive movement depends on cyclosis, it stops when cytoplasm movement is arrested with dinitrophenol (DNP) which blocks ATP synthesis.Rotation, however, persists for some time after DNP application which suggests that it is autonomous, independent of cytoplasm movement.Also the fact that peripheral speed of the rotating lipotubuloid reaches 31.4 μm/s and is 6.2 times faster than the maximum speed of cytoplasmic motion (Kwiatkowska, 1972a) proves the above suggestion.
The question arises what is the connection between microtubules and lipotubuloid movement.One thing seems certain, microtubules which join lipid bodies create one structure able to move as a unity despite not having its own membrane.Moreover, it turned out that microtubules of lipotubuloids differ in diameter (Fig. 14), two populations were revealed: wide (43-58 nm) and narrow (24-39 nm).In the lipotubuloids in the ovary epidermis which move less dynamically the number of wide microtubules is smaller (Kwiatkowska et al., 2006) than in the fast-moving lipotubuloids present in stipule (Kwiatkowska et al., 2009).The microtubule diameter depends on the varying number of protofilaments which form them, the bigger the number the greater the diameter (Fig. 15A).Regardless of the above correlation, analyses of microtubule cross-sections revealed that with the same number of filaments (e.g. 10, 11, 12) two microtubule populations were observed both in the control and after DNP removal while under DNP influence only one middle-sized population was present (Fig. 15B).It was also shown that the number of microtubule protofilaments in the control, under DNP influence and after its removal was stable.Analysing wall structure of microtubules varying in size but formed from the same number of protofilaments it was revealed that these changes depended on varying tubulin monomer sizes as well as different distances between them (Fig. 16).
Fig. 15.A -A scheme of microtubules whose width depends on the number of filaments; B -a graph presenting two microtubule populations in the control and after DNP removal, and one microtubule population after DNP application (Kwiatkowska et al., 2009).
In the wider microtubules both these parameters are greater and vice versa (Kwiatkowska et al., 2009).All the above proves flexibility of microtubules in vivo depending on their functional status.The fixation method used by us makes it possible to "freeze" the microtubule structure in the in vivo state due to quick OsO 4 penetration as was shown by Omoto & Kung (1980).Other authors observed microtubule flexibility in vitro (Nogales et al., 1999;Li et al., 2002;Pampaloni & Florin, 2008).
In O. umbellatum lipotubuloids, apart from microtubules, there are also short actin filaments which were observed in ultrastructural pictures (Fig. 3) (Kwiatkowska et al., 2005).A hypothesis has been put forward that interaction of actin filaments with microtubules may determine the transformation of wide microtubules into narrow ones and vice versa (Kwiatkowska et al., 2009).Microtubules of varying sizes were observed in vitro as a result of tubulin co-sedimentation in the presence of actin and myosin Va (Cao et al., 2004).We also suppose that changes in lipotubuloid microtubule sizes might the driving force of their autonomic rotation.
It is worth stressing that the rotary-progressive lipotubuloid movement plays an important role in substance exchange between them and a cell.This is supported by the results concerning the involvement of intracellular motion in spreading various substances in a cell (Verchot-Lubicz & Goldstein, 2010).

Microtubules and lipid synthesis in mature lipid bodies
Autoradiographic ultrastructural studies with the use of 3 H-palmitic acid showed that incorporation of this precursor into lipids took place at the site of microtubule adhesion to the half unit membrane (Fig. 11B-J) thus a hypothesis has been put forward that these two structures cooperate in lipid synthesis (Kwiatkowska, 2004).It is supported by the fact that after short radioactive incubation microtubules are labeled first while lipid bodies as late as after 2 h (Kwiatkowska et al., 2011b).Thus it can be assumed that microtubules take up lipid precursors, including radioactive particles, and transmit them to the incorporation site.
The immunogold labeling showed that gold grains, indicating the presence of two enzymes: DGAT1 and DGAT2 as well as of phospholipase D also indispensable for lipid synthesis (Andersson et al., 2006), were present at microtubule walls (Fig. 17, 18) (Kwiatkowska et al., 2011b).The results concerning phospholipase D correspond to these of the co-sedimentation assay in which microtubules decorated with phospholipase D were observed (Gardiner et al., 2001;Gardiner et al., 2003;Dhonukshe et al., 2003).
On the basis of autoradiography and immunogold labeling a hypothesis may be put forward that microtubules take an active part in lipid synthesis as transmitters of precursors and enzymes to their respective destinations.Valuable proofs of microtubule involvement in lipid synthesis come from research with their inhibitors.Pacheco et al. (2007) observed that colchicine or taxol similarly blocked lipid body formation, being the reaction to inflammation, in mouse monocytes.Recently a similar experiment has been carried out on O. umbellatum lipotubuloids, which were incubated for 6 h in propyzamide which is known to induce microtubule degradation (Nakamura et al., 2004;Sedbrook et al., 2004).It turned out that it induced partial microtubule disintegration and changed their structure by forming on their walls dark deposits visible in EM.Most probably they make microtubules lose their transmitting abilities, this leads to the blockade of new lipid synthesis which is reflected by inhibited incorporation of 3 H-palmitic acid into lipotubuloids.This seems to be the decisive proof of microtubule role in lipid synthesis (Kwiatkowska et al., 2011b).
We believe that in the case of lipotubuloids of other plants they may also function as transmitters of different substances to lipid bodies, however this issue needs further research.
Up till now there has been no research proving a similar role of microtubules in lipid synthesis in other organisms, although microtubules surrounding single lipid bodies, not organized into lipotubuloids, were observed in Marchantia paleacea (Galatis et al., 1978), Lactuca sativa (Smith, 1991) and in red alga Gelidium robustum (Delivopoulos, 2003).Small sizes and great lability of microtubules probably make observation of more common structural and functional correlation between microtubules and lipid bodies impossible.
As it was mentioned earlier lipotubuloids are rich in ribosomes and rough ER in the form of cisternae and vesicles.No detailed research concerning their functioning was conducted but it easily visible that ribosomes are actively involved in translation as they form numerous polysomes (Fig. 2).Since formation of new lipid bodies and lipid synthesis involve a whole enzymatic system and many regulatory factors active ER and ribosomes in lipotubuloids are indispensable.
Other structures such as mitochondria, microbodies surrounded with single lipid bi-layers (glyoxysomes and peroxysomes) as well as Golgi structures are less numerous and have not been studied in detail so far.It is believed that lipid bodies cooperate with other organelles as "gregarious" organelles (Goodman, 2008).Mitochondria and microbodies are in close contact with lipid bodies.Due to synaptic connections (Binns et al., 2006) there is correlation between release and oxidation of lipid acids resulting from lipolysis (Goodman, 2008;Fujimoto et al., 2008;Guo et al., 2009).Moreover, mitochondria may supply energy and NADPH which make lipid synthesis and lipid bodies biogenesis possible (Walter & Farese, 2009).
Golgi structures which are very dynamic organelles may be involved in microtubule polymerization as many authors believe (Chabin-Brion et al., 2001;Efimov et al., 2007;Kodani & Sutterlin, 2009).On the other hand, COPI (Beller et al., 2008) and COPII complexes (Soni et al., 2009) produced by Golgi structures are evolutionary conserved regulators of lipid homeostasis.At the final stage of lipotubuloid development autolytic vacuoles appear prior to microtubule disappearance leading to disintegration of lipotubuloids into separate lipid bodies (Fig. 6).These vacuoles are surrounded with a tonoplast and contain fragments of membranes and cell structures which is characteristic of autolytic vacuoles.Immunogold labeling revealed in them numerous gold grains indicating the presence of lipase (Fig. 19) (Kwiatkowska et al., in prep.).The above observation supports earlier results of cytochemical assays revealing lipase and acid phosphatase in lipotubuloids (Kwiatkowska, 1971b).Triggering of autolysis during dynamic metabolism was also observed in animal cells (Dong & Czaja, 2011).

Lipotubuloids and cuticle synthesis
It is known that cuticle are produced by epidermis cells which may dedicated more than 50% of their metabolites to this structure (Suh et al., 2005).In the case of O. umbellatum lipotubuloids incubated for 2 h in 3 H-palmitic acid and postincubated for 6 h in the nonradioactive medium autoradiographic grains first assembled over lipotubuloids become scattered all over a cell (tangential section) (Fig. 20).
After postincubation the number of autoradiographic grains falls by about 70% which means that a great amount of lipids was metabolized during 6 h.The remaining autoradiographic grains do not disappear after lipid extraction but their number drops (Tab. 2). Cell radioactive parts insoluble in solvents are visible on the epidermis cross section at the site corresponding to a cuticular layer from Bird's (2008) cuticule scheme (Fig. 21).Thus it seems very probable that there are cutins insoluble in lipid solvents.This part of scattered autoradiographic grains which disappeared after lipid extraction may correspond to waxes which are easily dissolved in organic solvents (Kwiatkowska et al., in prep.).A hypothesis has been put forward that about 30% of lipids from lipotubuloids turn into cuticle.A question arises if this transformation takes place in lipotubuloids or in other cell compartments and the lipids from lipotubuloids are only building blocks.This problem is worth elucidating since many recent results indicate its great importance with regard to economical and biotechnological issues (Heredia et al., 2009;Domίnguez et al., 2011).Cutin is the most ubiquitous biopolymer in biosphere (Heredia, 2003).We are planning to take up this question soon.

Conclusions
Lipotubuloids are a very specific, dynamic, complicated set of metabolically active (although seemingly static) lipid bodies containing DGAT and lipase which cooperate with microtubules and other organelles.A lipotubuloid is somewhat independent in a cell which is reflected by its capability for autonomous rotary movement, howev e r , i t i s c l o s e l y correlated with the development of the ovary epidermis and cuticle synthesis during seed formation in a fruit.Studies concerning these structures may further elucidate lipid metabolism and their functional relation with a cell and its organelles.

Fig. 11 .
Fig. 11.O. umbellatum lipotubuloid autoradiograms after 2 h 3 H-palmitic acid incorporation; A -light microscope picture of a lipotubuloid labeled with silver grains (arrows); B-Jelectronograms of silver grains localized at the half unit membrane/core border of lipid bodies (arrows); c -cytoplasm, l -lipotubuloid, lb -lipid bodies, mt -microtubules; bars: 10 μm (A), 0.2 μm (B-J),(Kwiatkowska et al., in prep.).Autoradiographic labeling disappears after lipid extraction with lipid solvents which shows that this precursor becomes incorporated into lipids.It is most clearly seen on light microscope pictures of autoradiograms where lipotubuloids are literally sprinkled with autoradiographic grains (Fig.11A) which disappear completely after lipid extraction.These pictures bring to the mind the term intuitively proposed by Wakker (1888) -"elaioplasts" which means -producing fat.

Fig. 12 .
Fig.12.Immunodetection of lipase in O. umbellatum ovary epidermis; A -Western blot analysis; line 1 -SDS-PAGE electrophoretic separation of the ovary epidermis extract; line 2 -Western blotting of the ovary epidermis extract probed with the anti-human lipase antibody; line 3 -molecular mass standards and their weights in kDa; B -lipid bodies with 20 nm gold grains indicating lipase presence; ER -endoplasmic reticulum, lb -lipid bodies, mt -microtubules; bar: 100 nm(Kwiatkowska et al., in prep.).

Fig. 13 .
Fig.13.A scheme of a lipotubuloid in an epidermal cell of O. umbellatum stipule which has turned around (several times) within 10-12 s, changing its direction and axis without a change in cellular location; c -cytoplasm, l -lipotubuloid, n -nucleus, v -vacuole, long arrows -the direction of lipotubuloid rotation, short arrows -direction of cytoplasm movement(Kwiatkowska et al., 2009).

Fig. 16 .
Fig. 16.Microtubules different in size consisting of the same number of protofilaments; visible differences in monomer sizes (arrows) and in distances between them; bars: 25 nm (Kwiatkowska et al., 2009).

Fig. 21
Fig. 21.A -Cross-section of O. umbellatum ovary epidermis (ep); autoradiogram after 2 h incubation in 3 H-palmitic acid, 6 h postincubation in the non-radioactive medium and after extraction in the lipid solvent; silver grains localized in the cuticular layer (Kwiatkowska et al., in prep.);B -a scheme of cuticle according to Bird (2008 -modified); bar: 5 μm.

Table 2 .
Number of silver grains over particular compartments of O. umbellatum ovary epidermis cell after incubation in 3 H-palmitic acid under different experimental conditions  SE.