David L. Gard

Organization, nucleation, and acetylation of microtubules in Xenopus laevis oocytes: a study by confocal immunofluorescence microscopy.

Anti-tubulin immunofluorescence and laser-scanning confocal microscopy were used to examine microtubule organization during Xenopus oogenesis (Dumont stages I-VI). Stage I oocytes contained a poorly ordered microtubule array, characterized by concentrations of microtubule in the cortex, surrounding the germinal vesicle, and associated with the mitochondrial mass. No focus of microtubule organization was detectable by optical sectioning or in microtubule regrowth experiments, suggesting that stage I oocytes lack a functional MTOC. The microtubule array becomes progressively more complex and polarized during oogenesis; an extensive array of microtubules and microtubule bundles was apparent in the animal hemisphere of stage VI oocytes, and a less ordered array was observed in the vegetal hemisphere. A dense network of microtubules surrounded the germinal vesicle, apparently extending from a tubulin- and microtubule-rich region of cytoplasm adjacent to the vegetal surface of the GV. The organization of microtubules in normal oocytes, in oocytes recovering from cold-induced microtubule depolymerization, and in enucleated oocytes, suggested that the germinal vesicle serves as an MTOC in stage VI oocytes. Antibodies to acetylated alpha-tubulin revealed numerous acetylated, presumably stable, microtubules in stage I and stage VI oocytes. The array of oocyte microtubules thus might function as a stable framework for the localization of developmentally important molecules and organelles during oogenesis.

Microtubule organization during maturation of Xenopus oocytes: Assembly and rotation of the meiotic spindles

Assembly of the meiotic spindles during progesterone-induced maturation of Xenopus oocytes was examined by confocal fluorescence microscopy using anti-tubulin antibodies and by time-lapse confocal microscopy of living oocytes microinjected with fluorescent tubulin. Assembly of a transient microtubule array from a disk-shaped MTOC was observed soon after germinal vesicle breakdown. This MTOC-TMA complex rapidly migrated toward the animal pole, in association with the condensing meiotic chromosomes. Four common stages were observed during the assembly of both M1 and M2 spindles: (1) formation of a compact aggregate of microtubules and chromosomes; (2) reorganization of this aggregate resulting in formation of a short bipolar spindle; (3) an anaphase-B-like elongation of the prometaphase spindle, transversely oriented with respect to the oocyte A-V axis; and (4) rotation of the spindle into alignment with the oocyte axis. The rate of spindle elongation observed in M1 (0.7 microns min-1) was slower than that observed in M2 (1.8 microns min-1). Examination of spindles by immunofluorescence with antitubulin revealed numerous interdigitating microtubules, suggesting that prometaphase elongation of meiotic spindles in Xenopus oocytes results from active sliding of antiparallel microtubules. A substantial number of maturing oocytes formed monopolar microtubule asters during M1, nucleated by hollow spherical MTOCs. These monasters were subsequently observed to develop into bipolar M1 spindles and proceed through meiosis. The results presented define a complex pathway for assembly and rotation of the meiotic spindles during maturation of Xenopus oocytes.

MAPping the eukaryotic tree of life: structure, function, and evolution of the MAP215/Dis1 family of microtubule-associated proteins.

The MAP215/Dis1 family of proteins is an evolutionarily ancient family of microtubule-associated proteins, with characterized members in all major kingdoms of eukaryotes, including fungi (Stu2 in S. cerevisiae, Dis1 and Alp14 in S. pombe), Dictyostelium (DdCP224), plants (Mor1 in A. thaliana and TMBP200 in N. tabaccum), and animals (Zyg9 in C. elegans, Msps in Drosophila, XMAP215 in Xenopus, and ch-TOG in humans). All MAP215/Dis1 proteins (with the exception of those in plants) localize to microtubule-organizing centers (MTOCs), including spindle pole bodies in yeast and centrosomes in animals, and all bind to microtubules in vitro and?or in vivo. Diverse roles in regulating microtubule assembly and organization have been proposed for individual family members, and a substantial body of evidence suggests that MAP215/Dis1-related proteins play critical roles in the assembly and function of the meiotic/mitotic spindles and/or cell division. An extensive search of public databases (including both EST and genome databases) identified partial sequences predicted to encode more than three dozen new members of the MAP215/Dis1 family, including putative MAP215/Dis1-related proteins in Giardia lamblia and four other protists, sixteen additional species of fungi, six plants, and twelve animals. The structure and function of MAP215/Dis1 proteins are discussed in relation to the evolution of this ancient family of microtubule-associated proteins.

Phosphorylation by CDK1 regulates XMAP215 function in vitro.

XMAP215, a microtubule-associated protein isolated from Xenopus eggs, promotes microtubule assembly dynamics in an end-specific manner: addition of XMAP215 to purified porcine tubulin increases both elongation and shortening rates at microtubule plus ends, with minimal effects at minus ends. Previous results indicated that XMAP215 is phosphorylated during M phase, suggesting that its activity may be regulated by cell cycle phosphorylation. To test this hypothesis, we used video-enhanced DIC microscopy to examine the effects of XMAP215 phosphorylated by CDK1 on the assembly of purified tubulin. XMAP215 incubated with ATP at 30 degrees C for 10- 20 min in the absence of CDK1 exhibited a 4.1-fold increase in plus end elongation rate compared to purified tubulin. Elongation was promoted to a lesser degree (2.4-fold) by phosphorylated XMAP215. In contrast, XMAP215 phosphorylation did not alter the approximately 3-fold increase in shortening rate. XMAP215 binding to taxol microtubules was also not changed by phosphorylation. To further investigate mechanisms responsible for the faster microtubule shortening rate observed with XMAP215, we made microtubules with segments assembled prior to XMAP215 addition (proximal segments) and segments assembled in the presence of XMAP215 (distal segments). In 9 of 10 microtubules, the distal segment shortened faster (distal = 60.7 microm/min; proximal = 37.5 microm/min), suggesting that MTs assembled in the presence of XMAP215 have an altered lattice that results in subsequent faster shortening.

F-actin is required for spindle anchoring and rotation in Xenopus oocytes: a re-examination of the effects of cytochalasin B on oocyte maturation

We used confocal immunofluorescence microscopy to examine spindle migration, morphology and orientation during the maturation of Xenopus oocytes, in the presence or absence of cytochalasin B (CB), an inhibitor of actin assembly. Treatment with CB during maturation (10-50 micrograms/ml beginning 0-3 h prior to addition of progesterone) disrupted the normal organisation of the novel MTOC and transient microtubule array (MTOC-TMA complex) that serves as the immediate precursor of the first meiotic spindle, suggesting that F-actin plays an important role in the assembly or maintenance of this complex. However, CB treatment did not block translocation of the MTOC-TMA complex to the oocyte cortex, suggesting that MTOC-TMA translocation is not dependent on an actin-based mechanism. Bipolar spindles were observed in CB-treated oocytes fixed during both M1 and M2. However, rotation of the M1 and M2 spindles into an orientation orthogonal to the oocyte surface was inhibited by CB. Rhodamine-phalloidin revealed a concentration of F-actin at the site of M1 spindle attachment, further suggesting that cortical actin is required for anchoring and rotation of the meiotic spindles. Finally, the incidence of M1 monasters was significantly increased in CB-treated oocytes, suggesting that interactions between the nascent M1 spindle and cortex are dependent on F-actin.

12 Confocal Immunofluorescence Microscopy of Microtubules, Microtubule-Associated Proteins, and Microtubule-Organizing Centers during Amphibian Oogenesis and Early Development

Publisher Summary During the past 5 years, the picture of the microtubule (MT) cytoskeleton of amphibian oocytes has changed dramatically, due in large part to refinements in the techniques for preserving and visualizing the oocyte cytoskeleton. Individual MTs are first identified in the cortex of Xenopus oocytes in samples prepared by rapid freezing. Subsequently, whole-mount immunocytochemistry and confocal immunofluorescence microscopy has revealed an extensive network of MTs extending throughout the cytoplasm of oocytes from both X. laevis and Rana pipiens . In addition, these techniques have revealed that dramatic changes in MT assembly, organization, and dynamics accompany distinct stages in oocyte differentiation, maturation, and early embryonic development. This chapter presents a view of the assembly and organization of the MT cytoskeleton during oogenesis and early development in the African frog, X. laevis , which is based on results obtained in laboratories around the world. Results obtained from other amphibian species are discussed when informative. In addition, some of the mechanisms that might serve to regulate MT organization and dynamics during amphibian oogenesis and early development are addressed, focusing on the potential contributions of microtubule organizing centers (MTOCs) and microtubule-associated proteins (MAPS).

Confocal microscopy and 3-D reconstruction of the cytoskeleton of Xenopus oocytes.

Xenopus oocytes contain a complex cytoskeleton composed of three filament systems: (1) microtubules, composed of tubulin and at least three different microtubule‐associated proteins (XMAPs); (2) microfilaments composed of actin and associated proteins; and (3) intermediate filaments, composed of keratins. For the past several years, we have used confocal immunofluorescence microscopy to characterize the organization of the oocyte cytoskeleton throughout the course of oogenesis. Together with computer‐assisted reconstruction of the oocyte in three dimensions, confocal microscopy gives an unprecedented view of the assembly and reorganization of the cytoskeleton during oocyte growth and differentiation. Results of these studies, combined with the effects of cytoskeletal inhibitors, suggest that organization of the cytoskeleton in Xenopus oocytes is dependent upon a hierarchy of interactions between microtubules, microfilaments, and keratin filaments. This article presents a gallery of confocal images and 3‐D reconstructions depicting the assembly and organization of the oocyte cytoskeleton during stages 0‐VI of oogenesis, a discussion of the mechanisms that might regulate cytoskeletal organization during oogenesis, and speculates on the potential roles of the oocyte cytoskeleton during oogenesis and axis formation. Microsc. Res. Tech. 44:388–414, 1999.

Confocal immunofluorescence microscopy of microtubules in amphibian oocytes and eggs.

Publisher Summary The advent and increasing availability of confocal microscopes has dramatically facilitated immunofluorescence microscopy of microtubule organization in large cells. The optical sectioning capabilities afforded by confocal microscopy eliminate the out-of-focus fluorescence that degrades conventional epifluorescence images of large cells and allows the rapid collection of images without the inconvenience of physical sectioning or complicated computerized deconvolution. This chapter focuses on the confocal immunofluorescence microscopy to examine microtubules organization in amphibian oocytes, eggs, and early embryos. The chapter provides a practical guide to immunofluorescence and confocal laser scanning microscopy (CLSM) of microtubules. The chapter compares the preservation of microtubules in oocytes fixed by several different protocols. Several problems were often encountered with cells fixed in methanol—significant shrinkage and distortion of oocytes, poor preservation of microtubules, and fragile oocytes. The most consistent preservation of individual microtubules throughout oogenesis and early embryogenesis was obtained with combinations of formaldehyde and glutaraldehyde. The chapter compares glutaraldehyde concentrations ranging from less than 0.1 to 2%.

Confocal microscopy of F-actin distribution in Xenopus oocytes.

We have used rhodamine-conjugated phalloidin and confocal microscopy to examine the organisation of filamentous actin (F-actin) during oogenesis in Xenopus laevis. F-actin was restricted to a thin shell in the cortex of oogonia and post-mitotic oocytes less than 35 microns in diameter. In oocytes with diameters of 35-50 microns, F-actin was observed in three cellular domains: in the cortex, in the germinal vesicle (GV) and in a network of cytoplasmic cables. Initially, actin cables were sparsely distributed in the cytoplasm, with no evidence of discrete organising centres. In larger stage I oocytes, a dense network of actin cables extended throughout the cytoplasm, linking the GV and mitochondrial mass to the cortical actin shell. Apart from the F-actin associated with the mitochondrial mass, no evidence of a polarised distribution of F-actin was apparent in stage I oocytes. F-actin was observed also in the cortex and the GV of stage VI oocytes, and a network of cytoplasmic cables surrounded the GV. Cytoplasmic actin cables extended from the GV to the animal cortex, and formed a three-dimensional network surrounding clusters of yolk platelets in the vegetal cytoplasm. Finally, disruption of F-actin in stage VI oocytes with cytochalasin resulted in distortion and apparent rotation of the GV in the animal hemisphere, suggesting that actin plays a role in maintaining the polarised organisation of amphibian oocytes.

Visualization of the cytoskeleton in Xenopus oocytes and eggs by confocal immunofluorescence microscopy.

Xelopus oocytes and eggs are popular models for studying the developmental and cellular mechanisms of RNA localization, axis specification and establishment, and nuclear envelope assembly/disassembly. However, their large size and opacity hamper application of many techniques commonly used for studying cell structure and organization, including immunofluorescence and other techniques of fluorescence microscopy. In this chapter, we describe techniques and procedures that we have used to preserve, stain, and view the cytoskeleton in Xenopus oocytes and eggs by confocal immunofluorescence microscopy.