Gamma synuclein: subcellular localization in neuronal and non-neuronal cells and effect on signal transduction.

Synucleins are small, highly conserved proteins in vertebrates, especially abundant in neurons and typically enriched in presynaptic terminals. alpha-Synuclein protein and a fragment of it, called NAC, have been found in association with pathological lesions of neurodegenerative diseases. Recently, mutations in a alpha-synuclein gene have been reported in families susceptible to an inherited form of Parkinson's diseases. In addition, alpha-synuclein has been implicated in the pathophysiology of other neurodegenerative diseases, including Alzheimer's disease and multiple system atrophy. Far less is known about other members of the synuclein family, beta- and gamma-synucleins. gamma-synuclein is up-regulated in several types of cancer and may affect the integrity of the neurofilament network, while its bovine ortholog, synoretin, activates the Elk-1 signal transduction pathway. In this paper, we present data about the localization and properties of human and bovine gamma-synuclein in several neuronal and non-neuronal cell cultures derived from ocular tissues. We show that gamma-synuclein is present in the perinuclear area and is localized to centrosomes in several types of human interphase cells and in bovine retinal pigment epithelium. In mitotic cells, gamma-synuclein staining is localized to the poles of the spindle. Further, overexpression of synoretin in retinoblastoma cells up-regulates MAPK and Elk-1. These results support the view that gamma-synuclein is a centrosome protein that may be involved in signal transduction pathways.

Reconstitution of centrosome microtubule nucleation in Spisula.

Evidence strongly supports the hypothesis that gamma-TuRCs are sites for the nucleation of microtubules within the centrosome PCM (Zheng et al., 1995; Moritz et al., 1995a,b). Further, these structures appear to be recruited to the centrosome from cytoplasmic pools during centrosome assembly and centrosome maturation (Moritz et al., 1998; Schnackenberg et al., 1998, 2000; Schnackenberg and Palazzo, 1999; Khodjakov and Rieder, 1999). It has also been shown that the centrosome PCM contains a network of filamentous fibers, which function as a scaffold for binding these microtubule nucleating sites (Schnackenberg et al., 1998). However, binding gamma-tubulin rings to this scaffold requires at least one additional factor (Moritz et al., 1998; Schnackenberg et al., 2000). Because extracts prepared from a variety of biological sources are capable of supporting the recovery of microtubule nucleation by Spisula KICRs (Schnackenberg et al., 2000), the methods outlined in this chapter could prove useful in the search for factors that are necessary for centrosome assembly and the increase in centrosome-dependent microtubule nucleation during centrosome maturation.

Kendrin/pericentrin-B, a centrosome protein with homology to pericentrin that complexes with PCM-1.

The centrosome is responsible for nucleating microtubules and performing other cellular roles. To define the organization of the centrosome more completely, a human anti-centrosome serum was used to screen a human cDNA library, and a cDNA encoding a >350 kDa centrosome protein was identified. Sequence analyses revealed that this novel centrosome protein contains two coiled-coil domains bounded by non-coiled regions. The N-terminal region of the protein, named pericentrin-B, shares 61% identity (75% similarity) with pericentrin, suggesting an evolutionary relationship between these proteins. Antibodies against pericentrin-B stain centrosomes at all stages of the cell cycle, and pericentrin-B remains associated with centrosomes following microtubule depolymerization. Immunodepletion of neither pericentrin-B nor PCM-1 from cellular extracts inhibited the ability of salt-stripped centrosomes to recover microtubule nucleation potential, demonstrating that neither protein plays a key role in microtubule nucleation processes. Moreover, the binding of both PCM-1 and pericentrin-B with salt-stripped centrosomes required intact microtubules, demonstrating that the association of PCM-1 and pericentrin-B with centrosomes is a late event in the centrosome maturation process. Finally, pericentrin-B and PCM-1 coimmunoprecipitate, suggesting that PCM-1 and pericentrin-B form a functional complex in cells. This observation may help to explain the generation of anti-centrosome autoantibodies in certain autoimmune patients and may be important for centrosome function.

In vitro reconstitution of fish melanophore pigment aggregation.

Movement and positioning of melanophore pigment organelles depend on microtubule- and actin-dependent motors, but the regulation of these forces are poorly understood. Here, we describe a cell free and fixed time motility assay for the study of the regulation of microtubule-dependent pigment organelle positioning in vitro. The assay involves introduction of microtubule-asters assembled in clam oocyte lysates into lysates prepared from Fundulus heteroclitus melanophores with either aggregated or dispersed pigment. When asters were introduced in lysates prepared from melanophores with dispersed pigment, pigment organelles bound astral microtubules and were evenly distributed throughout the aster. In contrast, when asters were introduced into lysates prepared from melanophores with aggregated pigment, pigment organelles accumulated around the centrosome, mimicking a pigment aggregate. The addition of anti-dynein intermediate chain antibody (m74-1), previously shown to interfere with binding of dynactin to dynein and thereby causing detachment of dynein from organelles, blocked the ATP-dependent aggregation of pigment in vitro and induced a depletion of pigment from the centrosomal area. The results show that dynein is essential for pigment aggregation and involved in maintenance of evenly dispersed pigment in vitro, analogous to cellular evidence, and suggest a possible role for dynactin in these processes as well.

Centrosome maturation.

In the past, centrosome maturation has been described as the change in microtubule nucleation potential that occurs as cells pass through specific phases of the cell cycle. It is suggested that the idea of centrosome maturation be expanded to include gain of functions that are not necessarily related to microtubule nucleation. Some of these functions could be transient and dependent on the temporary association of molecules with the centrosome as cells progress through the cell cycle. Thus, the centrosome may best be viewed as a site for mediating macromolecular interactions, perhaps as a central processing station within the cell. The centromatrix, a relatively stable lattice of polymers within the centrosome's PCM, could serve as a scaffold for the transient binding of mediator molecules, as well as allow the dynamic exchange of centrosome constituents with a soluble cytoplasmic pool. New evidence adds support to the idea that centrioles are crucial for the maintenance of PCM structure. However, significant evidence indicates that aspects of centrosome structure and function can be maintained in the absence of centrioles. In the case of paternal centrosome maturation, sperm centrioles may not contain an associated centromatrix. It is proposed that regulation of paternal centrioles or centriole associated proteins could mediate centriole-dependent centromatrix assembly following fertilization. Thus, regulation of centromatrix-centriole interactions could be involved in maintaining the integrity of the centrosome's PCM and play an important role in centrosome disassembly during cell differentiation and morphogenesis.

Reconstitution of microtubule nucleation potential in centrosomes isolated from Spisula solidissima oocytes.

Treatment of isolated Spisula solidissima centrosomes with KI removes (gamma)-tubulin, 25 nm rings, and their microtubule nucleation potential, revealing the presence of a filamentous lattice, the 'centromatrix'. Treatment of this centromatrix with Spisula oocyte extract results in the binding of (gamma)-tubulin and 25 nm rings, and the recovery of microtubule nucleation potential. Fractionation of this extract resulted in the separation of elements that are required for the recovery of microtubule nucleation potential. We show that some, but not all, of the elements needed cosediment with microtubules. Further, extracts prepared from activated (meiotic) and non-activated (interphase) Spisula oocytes, CHO cells blocked in S phase, Drosophila embryos and Xenopus oocytes all support the recovery of microtubule nucleation potential by the Spisula centromatrix. These results demonstrate that components necessary for centrosome-dependent microtubule nucleation are functionally conserved and abundant in both interphase and meiotic/mitotic cytoplasm.

Identification and function of the centrosome centromatrix.

Centrosomes direct the organization of microtubules in animal cells. However, in the absence of centrosomes, cytoplasm has the potential to organize microtubules and assemble complex structures such as anastral spindles. During cell replication or following fertilization, centrioles that are incapable of organizing microtubules into astral arrays are introduced into this complex cytoplasmic environment. These centrioles become associated with pericentriolar material responsible for centrosome-dependent microtubule nucleation, and thus the centrosome matures to ultimately become a dominant microtubule organizing center that serves as a central organizer of cell cytoplasm. We describe the identification of a novel structure within the pericentriolar material of centrosomes called the centromatrix. The centromatrix is a salt-insoluble filamentous scaffold to which subunit structures that are necessary for microtubule nucleation and abundant in the cytoplasm bind. We propose that the centromatrix serves to concentrate and focus these subunits to form the microtubule organizing center. Since binding of these subunits to the centromatrix does not require nucleotides, we propose a model for centrosome assembly which predicts that the assembly of the centromatrix is a rate-limiting step in centrosome assembly and maturation.

Dynein is required for spindle assembly in cytoplasmic extracts of Spisula solidissima oocytes.

Meiosis I spindle assembly is induced in lysate-extract mixtures prepared from clam (Spisula solidissima) oocytes. Unactivated lysate prepared from unactivated oocytes contain nuclei (germinal vesicles, GVs) which house condensed chromosomes. Treatment of unactivated lysate with clarified activated extract prepared from oocytes induced to complete meiosis by treatment with KCl induces GV breakdown (GVBD) and assembly of monopolar, bipolar, and multipolar aster-chromosome complexes. The process of in vitro meiosis I spindle assembly involves the assembly of microtubule asters and the association of these asters with the surfaces of the GVs, followed by GVBD and spindle assembly. Monoclonal antibody m74-1, known to react specifically with the N terminus of the intermediate chain of cytoplasmic dynein, recognizes Spisula oocyte dynein and inhibits in vitro meiosis I spindle assembly. Control antibody has no affect on spindle assembly. A similar inhibitory effect on spindle assembly was observed in the presence of orthovanadate, a known inhibitor of dynein ATPase activity. Neither m74-1 nor orthovanadate has any obvious affect on GVBD or aster formation. We propose that dynein function is required for the association of chromosomes with astral microtubules during in vitro meiosis I spindle assembly in these lysate-extract mixtures. However, we conclude that dynein function is not required for centrosome assembly and maturation or for centrosome-dependent aster formation.

Differential regulation of maternal vs. paternal centrosomes.

Centrosomes are the main microtubule-organizing centers in animal cells. During meiosis and mitosis, two centrosomes form the poles that direct the assembly of a bipolar spindle, thus ensuring the accurate segregation of chromosomes. Cells cannot tolerate the presence of more than two active centrosomes during meiosis or mitosis because doing so results in the formation of multipolar spindles, infidelity in chromosome segregation, and aneuploidy. Here, we show that fertilization of Spisula solidissima oocytes results in cells that contain three active centrosomes, two maternal and one paternal. During meiosis I, the paternal centrosome's ability to nucleate microtubules is selectively shut off while maternal centrosomes remain competent to nucleate microtubules and assemble asters in the same cytoplasm. We propose that embryos can identify paternal vs. maternal centrosomes and can control them differentially.

Isolation of centrosomes from Spisula solidissima oocytes.

We have described methods for the preparation of lysates and isolation of centrosomes from parthenogenetically activated oocytes of the surf clam, S. solidissima. Although oocyte availability is seasonal, between June and August as much as 2 liters of lysate can be generated by a single person. Since lysate can be stored frozen at -80 degrees C with no apparent loss in centrosome-dependent microtubule nucleation, this is a convenient system for year-round experimentation. On average, per milliliter of frozen-stored lysate, 2 or 3 x 10(6) centrosomes can be obtained at 3000- or 4000-fold purification by sucrose-density gradient centrifugation. Centrosome fractions typically contain 6.0 x 10(-12) g of protein per centrosome (Vogel et al., 1997) and 140-200 micrograms of protein is usually obtained from a single run involving six sucrose-density gradients (12 ml of lysate). One person can easily run three preparations in a day, and thus 420-600 micrograms of centrosome protein could be prepared daily. Therefore, based on the effort of one individual, as much as 20-40 mg of centrosome protein could be prepared per year. Another convenient feature of the system is that once centrosomes are isolated, they can be stored in high sucrose media at -80 degrees C for years with little or no loss in microtubule nucleation potential. Once isolated, centrosomes can be used for protein analysis, ultrastructural studies, or in functional reconstitution assays (Vogel, 1997). In addition, these preparations offer the isolation of sufficient quantities of centrosome proteins to be used as antigens for generating centrosome-specific antibodies or for obtaining protein sequence for the purpose of antibody production or the design of oligonucleotide primers for isolating cDNA fragments coding for centrosome proteins. Thus, the preparations described offer a biochemical approach for defining centrosome composition. The methods described for immunofluorescence analysis of asters assembled in lysates offer rapid and convenient preparations for screening antibodies for centrosome localization and specificity. Finally, the ability to prepare large quantities of homogeneous centrosomes should enhance ultrastructural studies since many centrosomes can be sectioned and analyzed simultaneously by EM, avoiding the problem of having to hunt through sections of single cells to find a single centrosome for analysis. In addition, colloidal gold localization studies, using antibodies and EM to pinpoint the relative location of individual proteins, could be carried out on populations of centrosomes in the same preparation simultaneously, thus drastically expanding the quantity of data gathered. In conclusion, the clam oocyte system described here offers the potential for a combined structural and biochemical approach for identification of novel centrosome proteins and elucidation of the molecular basis of microtubule nucleation, centrosome assembly, and centrosome function.

The disassembly and reassembly of functional centrosomes in vitro.

Animal cells contain a single centrosome that nucleates and organizes a polarized array of microtubules which functions in many cellular processes. In most cells the centrosome is composed of two centrioles surrounded by an ill-defined "cloud" of pericentriolar material. Recently, gamma-tubulin-containing 25-nm diameter ring structures have been identified as likely microtubule nucleation sites within the pericentriolar material of isolated centrosomes. Here we demonstrate that when Spisula centrosomes are extracted with 1.0 M KI they lose their microtubule nucleation potential and appear by three-dimensional electron microscopy as a complex lattice, built from 12- to 15-nm thick elementary fiber(s), that lack centrioles and 25-nm rings. Importantly, when these remnants are incubated in extracts prepared from Spisula oocytes they recover their 25-nm rings, gamma-tubulin, and microtubule nucleation potential. This recovery process occurs in the absence of microtubules, divalent cations, and nucleotides. Thus, in animals the centrosome is structurally organized around a KI-insoluble filament-based "centromatrix" that serves as a scaffold to which those proteins required for microtubule nucleation bind, either directly or indirectly, in a divalent cation and nucleotide independent manner.

Centrosomes isolated from Spisula solidissima oocytes contain rings and an unusual stoichiometric ratio of alpha/beta tubulin.

Centrosome-dependent microtubule nucleation involves the interaction of tubulin subunits with pericentriolar material. To study the biochemical and structural basis of centrosome-dependent microtubule nucleation, centrosomes capable of organizing microtubules into astral arrays were isolated from parthenogenetically activated Spisula solidissima oocytes. Intermediate voltage electron microscopy tomography revealed that each centrosome was composed of a single centriole surrounded by pericentriolar material that was studded with ring-shaped structures approximately 25 nm in diameter and <25 nm in length. A number of proteins copurified with centrosomes including: (a) proteins that contained M-phase-specific phosphoepitopes (MPM-2), (b) alpha-, beta-, and gamma-tubulins, (c) actin, and (d) three low molecular weight proteins of <20 kD. gamma-Tubulin was not an MPM-2 phosphoprotein and was the most abundant form of tubulin in centrosomes. Relatively little alpha- or beta-tubulin copurified with centrosomes, and the ratio of alpha- to beta-tubulin in centrosomes was not 1:1 as expected, but rather 1:4.6, suggesting that centrosomes contain beta-tubulin that is not dimerized with alpha-tubulin.

Analysis of the calcium transient at NEB during the first cell cycle in dividing sea urchin eggs.

Accumulating evidence from several systems suggests that nuclear envelope breakdown (NEB) is triggered by an endogenous transient of free calcium. Using h- and f-semisynthetic aequorins as cytosolic calcium indicators, we have clearly and regularly visualized a single large, global calcium transient just before first NEB in normally developing, monospermic Lytechinus eggs. Although similar transients were not observed at NEB in subsequent cell cycles, microinjection of the calcium buffer BAPTA into one blastomere of the two-celled embryo resulted in the inhibition of NEB. The NEB transient in the first cell cycle was some five-fold smaller than the one associated with egg activation. Our data suggest that this transient takes the form of a calcium wave that spreads inwards from the periphery of the egg toward the nucleus. We confirmed that these NEB transients did not require extracellular Ca2+. In polyspermic eggs, NEB-associated transients were four-fold larger than in monospermic eggs and were periodically repeated. Examination of the distribution of fluorescein-conjugated aequorins with a laser scanning confocal microscope indicated that aequorin both enters the nucleus and is evenly distributed within the cytosol of the egg. The use of h- and f-aequorins did not reveal any NEB transients during subsequent cell cycles, nor did we detect transients associated with other cell cycle events. However, a complex train of calcium transients in the form of both localized pulses and propagated waves was detected from embryos beginning at about the morula-to-blastula transition and continuing through to hatching.

Sperm nuclear transformations in cytoplasmic extracts from surf clam (Spisula solidissima) oocytes.

Following their incorporation into oocytes, sperm nuclei (SN) of the surf clam, Spisula solidissima, undergo an initial expansion, followed by condensation and then a dramatic enlargement during their development into male pronuclei. These changes are temporally correlated with alterations in the maternal chromatin: germinal vesicle breakdown (GVBD), meiotic maturation, and female pronuclear development, respectively. To analyze possible changes occurring in SN at fertilization, surf clam oocyte extracts, prepared before and after parthenogenetic activation, were examined for their ability to affect SN in vitro. Sperm heads were incubated in extracts for variable periods up to 5 hr. Extracts prepared from oocytes following GVBD (15 min postactivation) induced an expansion in approximately 90% of SN by 60 min incubation. However, when SN were incubated in extracts from unactivated or 4-min-activated oocytes only approximately 30% underwent expansion. Ultrastructural examination of specimens taken at increasing periods of incubation in oocyte extracts revealed that SN expansion in vitro resembled chromatin decondensation in vivo. SN incubated 1 to 5 hr in extracts prepared from oocytes following GVBD consisted of decondensed chromatin surrounded to varying degrees by membranous cisternae. Staining with anti-lamin antibody was variable: some specimens (60-70%) were positive while others (30-40%) were weak to negative. In contrast, all decondensed SN incubated in extracts from postmeiotic oocytes (65 min postactivation) were delimited by an intact nuclear envelope possessing nuclear pores and reactive to anti-lamin antibody. Decondensation of SN in 15- or 65-min extracts was blocked by EDTA, 2,6-dimethyl-ami-nopurine, histone, and protamine. The presence (65-min extract) and absence (unactivated, 4- and 15-min extracts) of sperm nuclear envelope assembly in vitro is consistent with events in vivo, where such a structure forms after meiotic maturation in concert with the development of the female pronucleus.

Preferential dendritic localization of pericentriolar material in hippocampal pyramidal neurons in culture.

Centrosomes are unique cytoplasmic structures which serve as microtubule organizing centers (MTOC). In most animal cells centrosomes consist of one or more pair of centrioles surrounded by electron dense amorphous pericentriolar material (PCM) responsible for nucleation of microtubules. In the present study we analyzed the pattern of induction and localization of proteins of the PCM at different stages of neuronal development in cell cultures prepared from the embryonic hippocampus. For this purpose we used a human polyclonal antibody that recognizes two proteins of the PCM (100 kd and 60 kd, respectively). The results indicate that in mature neurons, pericentriolar immunoreactive material is preferentially localized in dendritic processes, and that throughout the course of neurite development and differentiation it is systematically excluded from the neuron's axon. Western blot analysis showed that during neuronal development in situ, there is an increase in the immunoreactivity for both proteins recognized by this antibody. In contrast, in hippocampal pyramidal neurons that develop in culture, there is an increase in the 60 kd polypeptide, while the 100 kd one is not detected after 7 days in vitro.

Centriole duplication in lysates of Spisula solidissima oocytes.

A cell-free system has been developed that executes centriole duplication. Surf clam (Spisula solidissima) oocytes, arrested at late prophase of meiosis I, do not contain centrioles, centrosomes, or asters. Serial section high-voltage electron microscopy (HVEM) of asters and spindles isolated from potassium chloride-activated oocytes indicates that within 4 minutes oocytes assemble a single centriole that is duplicated by 15 minutes when assembly of the first meiotic spindle is complete. A mixture of lysates from unactivated oocytes and potassium chloride-activated oocytes induces centriole formation and duplication. Astral microtubule content in these lysate mixtures increases with time.

Regulation of M-phase progression in Chaetopterus oocytes by protein kinase C.

We have examined the presence of protein kinase C in oocytes of Chaetopterus pergamentaceus and its role in the initiation of germinal vesicle breakdown (GVBD). First, we demonstrated that the oocytes contain a phospholipid- and calcium-dependent protein kinase, protein kinase C (PKC). Since PKC is the primary intracellular receptor for phorbol esters, we tested the ability of phorbol 12,13-dibutyrate (PDBu) to induce GVBD and compared several critical events and processes involved in GVBD induced by PDBu to those induced normally (by seawater). Seawater and 100-200 nM PDBu induced chromosome condensation, spindle formation, and spindle migration over a similar time course. Both treatments induced similar alterations in the SDS-PAGE pattern of newly synthesized proteins. The synthesis of polypeptides of approximately 46 and 54 kDa increased specifically. Both treatments increased oocyte protein phosphorylation, especially of proteins of 22, 32, 46, 55, 64, and 84 kDa. Both treatments resulted in the activation of an M-phase-specific histone H1 kinase activity, which demonstrates the appearance of maturation-promoting factor. Staurosporine, a potent protein kinase C inhibitor, blocked GVBD and the activation of M-phase-specific H1 kinase, whereas HA1004, which preferentially antagonizes protein kinase A, had no effect. The results of this study demonstrate that protein kinase C can activate a wide spectrum of essential biochemical and morphological processes involved in GVBD. Further, these studies suggest that protein kinase C elicits GVBD by activating maturation-promoting factor and support the hypothesis that protein kinase C plays an essential role in oocyte maturation in this species.