Antonio Tiezzi

The kinesin-immunoreactive homologue from Nicotiana tabacum pollen tubes: Biochemical properties and subcellular localization

In plant cells, microtubule-based motor proteins have not been characterized to the same degree as in animal cells; therefore, it is not yet clear whether the movement of organelles and vesicles is also dependent on the microtubular cytoskeleton. In this work the kinesinimmunoreactive homologue from pollen tubes of Nicotiana tabacum L. has been purified and biochemically characterized. The protein preparation mainly contained a polypeptide with a relative molecular weight of approx. 100 kDa. This polypeptide bound to animal microtubules in an ATP-dependent manner and it further copurified with an ATPase activity fourfold-stimulated by the presence of microtubules. In addition, the sedimentation coefficient (approx. 9S) was similar to those previously shown for other kinesins. Immunofluorescence analyses revealed a partial co-distribution of the protein with microtubules in the pollen tube. These data clearly indicate that several properties of the kinesin-immunoreactive homologue are similar to those of kinesin proteins, and suggest that molecular mechanisms analogous to those of animal cells may drive the microtubule-based motility of organelles and vesicles in plants.

Organization, Composition, and Function of the Generative Cell and Sperm Cytoskeleton

Publisher Summary This chapter focuses on the organization, composition, and function of the generative cell and sperm cytoskeleton. Microtubule (MT) organization in generative and sperm cells (GSP) contrasts sharply with that in the surrounding vegetative cytoplasm. MT is composed of heterodimers of α - and β -tubulin, which share sequence homology at the DNA and protein levels with counterparts in animals. Vegetative MTs in hydrated pollen grains and pollen tubes do not occur in similar thick bundles but instead comprise much thinner elements organized in various patterns. In pollen tubes MTs are arranged primarily in helical to longitudinal arrays in the outer cortex. The cortical elements consist of single MTs or planar groups that are cross-bridged to each other and to the plasma membrane. The MT bundles in GSP are also unlike somatic cell arrays. While cortical MTs are linked to the plasma membrane and associate with each other in somatic cells, the kind of large, flexuous bundles that ramify through the cytoplasm of GSP are not observed. Instead, the MTs are largely limited to a single layer next to the plasma membrane.

Identification and Characterization of a Novel Microtubule-Based Motor Associated with Membranous Organelles in Tobacco Pollen Tubes

Pollen tube growth depends on the differential distribution of organelles and vesicles along the tube. The role of microtubules in organelle movement is uncertain, mainly because information at the molecular level is limited. In an effort to understand the molecular basis of microtubule-based movement, we isolated from tobacco pollen tubes polypeptides that cosediment with microtubules in an ATP-dependent manner. Major polypeptides released from microtubules by ATP (ATP-MAPs) had molecular masses of 90, 80, and 41 kD. Several findings indicate that the 90-kD ATP-MAP is a kinesin-related motor: binding of the polypeptide to microtubules was enhanced by the nonhydrolyzable ATP analog AMP-PNP; the 90-kD polypeptide reacted specifically with a peptide antibody directed against a highly conserved region in the motor domain of the kinesin superfamily; purified 90-kD ATP-MAP induced microtubules to glide in motility assays in vitro; and the 90-kD ATP-MAP cofractionated with microtubule-activated ATPase activity. Immunolocalization studies indicated that the 90-kD ATP-MAP binds to organelles associated with microtubules in the cortical region of the pollen tube. These findings suggest that the 90-kD ATP-MAP is a kinesin-related microtubule motor that moves organelles in the cortex of growing pollen tubes.

DISTRIBUTION OF MICROTUBULES DURING THE GROWTH OF TOBACCO POLLEN TUBES

Summary— The distribution of microtubules was investigated in Nicotiana tabacum pollen tubes at different stages of tube growth by immunofluorescence microscopy. Using specific antibodies, the presence of microtubules consisting of different tubulin isoforms was tested. α‐, β‐ and tyrosinated α‐tubulin were present within the tube, whereas the acetylated form was lacking. The presence of tubulin subunits in pollen tube extracts was also investigated by immunoblotting analyses. The use of a confocal laser scanning microscope integrated with computer‐assisted imaging, allowed a detailed visualization of the microtubule distribution and organization. Cytoplasmic microtubules organized as short bundles with various orientations were detected at the apex of long tubes.

The pollen tube cytoskeleton

In the last few years the role of pollen and the pollen tube in the fertilization process in higher plants has received considerable attention. By ultrastructural, biochemical and immunofluorescent investigations it has been shown that a cytoskeletal apparatus plays a central role in pollen tube growth. Microfilaments and microtubules, in which main components are, respectively, actin and tubulin, represent the most investigated cytoskeletal components. New information has been recently provided by the identification of myosin and also of a kinesin-like protein. The pollen tube cytoskeleton consists of two different cytoskeletal systems: the vegetative cell cytoskeleton, namely the cytoskeleton of the pollen grain and pollen tube, and the gamete cytoskeleton (generative cell and sperm cell cytoskeleton). The vegetative cell cytoskeleton plays a fundamental role in assuring the cytoplasmic movement of organelles, vesicles and gametes from the pollen grain to the pollen tube apex and consists mainly of microtubules and microfilaments. Also myosin and the kinesin-like protein are involved in the process of organelle and vesicle movement. The gamete cytoskeleton has a central role in sperm cell formation and in the reshaping process during gamete movement inside the pollen tube. It consists mostly of microtubules and partially characterized microtubule-associated structures. Actin filaments have recently also been identified.

Confocal image analysis of spatial variations in immunocytochemically identified calmodulin during pollen hydration, germination and pollen tube tip growth in Nicotiana tabacum L.

Using monoclonal anti-calmodulin antibodies in conjunction with confocal scanning laser microscopy we have analysed the spatial variations in the distribution pattern of calmodulin (CaM) during the sequential events of pollen hydration, germination and tube growth in Nicotiana tabacum. These immunocytochemical observations have been complemented by immunochemical studies wherein the anti-calmodulin antibody raised against pea CaM recognises a polypeptide of c. 18 kDa in the pollen extracts. Digitisation of confocally acquired optical sections of immunofluorescence images reveals that in hydrated pollen a high level of CaM is consistently present in the region of the germinal apertures. Subsequently, with the onset of germination a high CaM concentration was found associated with the plasma membrane of the germination bubble and in the cytoplasm in its vicinity, while in the vegetative cytoplasm a weak diffuse and intense punctate signal was registered. CaM immunostain was also detected in association with the plasma membrane of the tube tips in both short and long pollen tubes. Furthermore, the cytosol of the tubes invariably manifested an apically focused CaM gradient. We were, however, unable to detect any vacuolar association of CaM in the older regions of the pollen tubes. Although punctate immunostain was obvious across the pollen tube numerous punctate structures were invariably present in the extreme tip. The possible implications of these findings in development of cell polarity, polarised growth, maintenance of calcium homeostasis and CaM interactions with other mechanochemical motor proteins in effecting propulsion of organelles during pollen hydration, germination and pollen tube growth are discussed.

Video microscopic observations of living, isolated embryo sacs of Nicotiana and their component cells

SummaryLiving embryo sacs and megagametophytic cells of Nicotiana alata and Nicotiana tabacum were obtained using enzymatic maceration and microdissection. The yields of isolated embryo sacs, egg apparatus and central cells were up to 35%, 40% and 35%, respectively. Vectorial movement of organelles and undulations of tubular structures, presumably endoplasmic reticulum, were observed in eggs, synergids and central cells using video-enhanced microscopy. Despite evident viability using the fluorochromatic reaction, the egg displays much less organelle movement and therefore appears to be quiescent. The large vacuole of the central cell is traversed by mobile strands of cytoplasm through which organelles migrate. A polygonal network is located at the periphery of the central cell, which may contribute to anchorage of the cell with the embryo-sac wall. The observation of organelle movement provides direct evidence of the condition of the cell and may be a useful approach for assessing cell vigor.

Antiproliferative activity of lichen extracts on murine myeloma cells

In the present study we report some preliminary results concerning the evaluation of antiproliferative activity on murine myeloma cells (P3X63-Ag8.653) of crude extracts of two common lichen species, Evernia prunastri and Xanthoria parietina.The results were evaluated by means of the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] test, which is commonly used to assess the activity of living cells through mitochondrial dehydrogenases. They indicated that extracts of E. prunastri had no effect, while those of X. parietina significantly affected murine myeloma cell proliferation, with a reduction down to 75% for methanolic extracts. This opens perspectives for deeper investigations extended also to other mammalian cell lines.

Ultrastructure of the stylar canal cells ofCitrus limon (Rutaceae)

The ultrastructure of the canal cells and the canal filling substance ofCitrus limon have been studied. At maturity the canal cells are very rich in cytoplasm. Their inner tangential walls lining the canal are much thickened and formed by two layers: the outer corresponds to the original wall, the inner is formed by subsequent deposition of abundant materials of different origin. This thickening occurs at the same time as the filling of the stylar canal. Both events are paralleled by considerable dictyosomic activity, the formation of a large amount of rough endoplasmic reticulum, and the incorporation of small cytoplasmic masses into the cell wall, due to plasmalemma evaginations. — The material in the stylar canal has a heterogeneous ultrastructure aspect and consists of polysaccharides, proteins and lipids; it presumably provides nutrients for the growing pollen tubes.

The anti-centrosome monoclonal antibody 6C6 reacts with a plasma membrane-associated polypeptide of 77 kDa from Nicotiana tabacum pollen tubes

SummaryIn the pollen and pollen tube of higher plants, the distribution of the microtubular cytoskeleton has been extensively studied. Even though the pattern of microtubules is known, one of the most remarkable deficiencies is the absence of data on the localization of microtubule-nucleation sites in the pollen tubes. In order to get insights about the localization of centrosome-like structures in the pollen tube ofNicotiana tabacum L., we have used the monoclonal antibody 6C6 to search for pericentriolar antigen(s). The antibody was initially raised against a component of animal centrosomes and has been already employed to locate centrosomal structures in other plant cell types. By immunoblotting analysis, a polypeptide of Mr 77,000 was identified specifically in the membrane-associated protein fraction of the pollen tube, and is absent from the soluble protein pool. Immunofluorescence observations have shown the polypeptide to be located in the apical part of the pollen tube (about 40–50 μm from the tip) in association with the cortical area. A purified plasma membrane fraction from the growing pollen tubes has been obtained, using H+-ATPase activity as an organelle marker. The plasma membrane fraction was shown to be enriched in the Mr 77,000 polypeptide, which can be extracted from membranes by treatment with the detergent CHAPS at a concentration of 0.5%. These data open new research perspectives on the localization and analysis of putative cortical microtubule nucleation sites in the pollen tube.