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PubMed2001

Systematic evolution of a DNA aptamer binding to rat brain tumor microvessels. selective targeting of endothelial regulatory protein pigpen.

Tumor microvessels differ in structure and metabolic function from normal vasculature, and neoangiogenesis is associated with quantitative and qualitative changes in expression of endothelial proteins. Such molecules could serve as molecular addresses differentiating the tumor vasculature from those of the normal brain. We have applied Systematic Evolution of Ligands by EXponential enrichment (SELEX) against transformed endothelial cells as a complex target to select single-stranded DNA-ligands (aptamers) that function as histological markers to detect microvessels of rat experimental glioma, a fatal brain tumor that is highly vascularized. Both the SELEX selection procedure as well as subsequent deconvolution-SELEX were analyzed by fluorescence based methods (flow cytometry and fluorescence microscopy). Of 25 aptamers analyzed, one aptamer was selected that selectively bound microvessels of rat brain glioblastoma but not the vasculature of the normal rat brain including peritumoral areas. The molecular target protein of aptamer III.1 was isolated from endothelial cells by ligand-mediated magnetic DNA affinity purification. This protein was identified by mass spectrometry as rat homologue of mouse pigpen, a not widely known endothelial protein the expression of which parallels the transition from quiescent to angiogenic phenotypes in vitro. Because neoangiogenesis, the formation of new blood vessels, is a key feature of tumor development, the presented aptamer can be used as a probe to analyze pathological angiogenesis of glioblastoma. The presented data show that pigpen is highly expressed in tumor microvessels of experimental rat brain glioblastoma and may play an important role in warranting blood supply, thus growth of brain tumors.

Animals↗
PubMed1981

Material from the internal surface of squid axon exhibits excess noise. Implications in modeling membrane noise.

A fluid material from a squid (Loligo pealei) axon was isolated by mechanical application of two types of microcapillary (1-3-mum Diam) to the internal surface of intact and cut-axon preparations. Current noise in the isolated material exceeded thermal levels and power spectra were 1/f in form in the frequency range 1.25-500 Hz with voltage-dependent intensities that were unrelated to specific ion channels. Whether conduction in this material is a significant source of excess noise during axon conduction remains to be determined. Nevertheless, a source of excess noise external to or within an ion channel may not be properly represented solely as an additive term to the spectrum of ion channel noise; a deconvolution of these spectral components may be required for modeling purposes.

Animals↗
PubMed2001

Calcium signaling mechanisms in dedifferentiated cardiac myocytes: comparison with neonatal and adult cardiomyocytes.

Our studies focused on calcium sparking and calcium transients in cultured adult rat cardiomyocytes and compared these findings to those in cultured neonatal and freshly isolated adult cardiomyocytes. Using deconvolution fluorescence microscopy and spec trophotometric image capture, sequence acquisitions were examined for calcium spark intensities, calcium concentrations and whether sparks gave rise to cell contraction events. Observations showed that the preparation of dedifferentiated cardiomyocytes resulted in stellate, neonatal-like cells that exhibited some aspects of calcium transient origination and proliferation similar to events seen in both neonatal and adult myocytes. Ryanodine treatment in freshly isolated adult myocytes blocked the calcium waves, indicating that calcium release at the level of the sarcoplasmic reticulum and t-tubule complex was the initiating factor, and this effect of ryanodine treatment was also seen in cultured-dedifferentiated adult myocytes. However, experiments revealed that in both neonatal and cultured adult myocytes, the inositol triphosphate pathway (IP3) was a major mechanism in the control of intracellular calcium concentrations. In neonatal myocytes, the nucleus and regions adjacent to the plasma membrane we re major sites of calcium release and flux. We conclude: (1) culturing of adult cardiomyocytes leads them to develop mechanisms of calcium homeostasis similar in some aspects to those seen in neonatal cardiomyocytes; (2) neonatal myocytes rely on both extracellular and nuclear calcium for contractile function; and (3) freshly isolated adult myocytes use sarcoplasmic reticulum calcium stores for the initiation of contractile function.

Age Factors↗
PubMed1989

Regional pulmonary transit times in humans.

We measured the frequency distribution of erythrocyte (RBC) transit times in resected lobes of lungs in eight human subjects undergoing thoracotomy for peripheral lung tumors. RBC transit times were measured by the injection of radiolabeled blood flow and volume markers, which were counted in samples from the resected lung. In five of these subjects, the measurements from the resected lung were compared with preoperative measurements of the transit times of radiolabeled RBCs with a gamma camera-computer system. Time-activity curves from the cardiac chambers and the lung or its regions were obtained from which transit times were calculated by the centroid and deconvolution techniques. The reproducibility of transit times measured by this technique was assessed in another eight normal subjects, after sequential bolus injections of radiolabeled cells. The mean transit time of the upper lung region was longer (5.1 +/- 0.5 s) than that of the lower (4.1 +/- 0.6 s, P less than 0.05) in the preoperative study. Similarly, the mean transit time of the upper lung slice was longer (5.5 +/- 0.3 s) than that of the lower slice (3.8 +/- 0.3 s, P less than 0.05) in the resected lung specimens. We conclude that there was good agreement between these techniques and that there are long transit times in the upper regions of human lungs.

Blood Circulation Time↗
PubMed2006

Reversible kinetic analysis of Myc targets in vivo provides novel insights into Myc-mediated tumorigenesis.

Deregulated expression of the Myc transcription factor is a frequent causal mutation in human cancer. Thousands of putative Myc target genes have been identified in in vitro studies, indicating that Myc exerts highly pleiotropic effects within cells and tissues. However, the complexity and diversity of Myc gene targets has confounded attempts at identifying which of these genes are the critical targets mediating Myc-driven tumorigenesis in vivo. Acute activation of Myc in a reversibly switchable transgenic model of Myc-mediated beta cell tumorigenesis induces rapid tumor onset, whereas subsequent Myc deactivation triggers equally rapid tumor regression. Thus, sustained Myc activity is required for tumor maintenance. We have used this reversibly switchable kinetic tumor model in combination with high-density oligonucleotide microarrays to develop an unbiased strategy for identifying candidate Myc-regulated genes responsible for maintenance of Myc-dependent tumors. Consistent with known Myc functions, some Myc-regulated genes are involved in cell growth, cycle, and proliferation. In addition, however, many Myc-regulated genes are specific to beta cells, indicating that a significant component of Myc action is cell type specific. Finally, we identify a very restricted cadre of genes with expression that is inversely regulated upon Myc activation-induced tumor progression and deactivation-induced tumor regression. By definition, such genes are candidates for tumor maintenance functions. Combining reversibly switchable, transgenic models of tumor formation and regression with genomic profiling offers a novel strategy with which to deconvolute the complexities of oncogenic signaling pathways in vivo.

Animals↗
PubMed2000

Colocalization of multiple GABA(A) receptor subtypes with gephyrin at postsynaptic sites.

Clustering of gamma aminobutyric acid (GABA)(A) receptors to postsynaptic sites requires the presence of both the gamma2 subunit and gephyrin. Here, we analyzed by double-immunofluorescence staining the colocalization of gephyrin and major GABA(A)-receptor subtypes distinguished by the subunits alpha1, alpha2, alpha3, or gamma2 in adult rat brain. By using confocal laser scanning microscopy, GABA(A)-receptor subunit staining revealed brightly stained clusters that were colocalized with gephyrin-positive clusters of similar size and distribution in several brain regions, including cerebellum, hippocampus, thalamus, and olfactory bulb. In addition, a diffuse staining was observed for GABA(A)-receptor subunits in the neuropil, presumably representing extrasynaptic receptors. Overall, only few gephyrin-positive clusters were not colocalized with GABA(A)-receptor subunit clusters. Electron microscopic analysis in cerebellar cortex confirmed the selective postsynaptic localization of gephyrin. High-resolution images (voxel size, 50 x 50 x 150 nm) were restored with an iterative image deconvolution procedure based on a measured point-spread function to analyze the colocalization between GABA(A)-receptor subunits and gephyrin in individual clusters. This analysis revealed a considerable heterogeneity in the micro-organization of these presumptive GABAergic postsynaptic sites. For instance, whereas gephyrin- and gamma2 subunit-positive clusters largely overlapped in the cerebellar molecular layer, the colocalization was only partial in glomeruli of the granule cell layer, where small gephyrin clusters typically were "embedded" in larger GABA(A)-receptor clusters. These findings show that gephyrin is associated with a majority of GABA(A)-receptor subtypes in brain, and document the usefulness of image deconvolution for analyzing the structural organization of the postsynaptic apparatus by fluorescence microscopy.

Animals↗
PubMed1995

Amplified nocturnal luteinizing hormone (LH) secretory burst frequency with selective attenuation of pulsatile (but not basal) testosterone secretion in healthy aged men: possible Leydig cell desensitization to endogenous LH signaling--a clinical research center study.

The specific mechanisms underlying the relative hypogonadism of aging remain to be elucidated fully. We used frequent venous sampling (every 2.5 min), sensitive and specific LH and testosterone assays, and deconvolution analysis of the endocrine time series to delineate the differences between healthy young (n = 10, age 21-34 yr) and aged (n = 8, age 62-74 yr) men in the nocturnal secretion of LH and testosterone and their half-lives. We found that elderly men vs. young men had more frequent bursts of LH secretion (1.4 vs. 0.9/h, P = 0.003), less testosterone secreted per testosterone secretory burst (9.2 vs. 17.0 nmol/L, P = 0.021), and less testosterone secreted per hour (10.7 vs. 25.0 nmol/L.h, P = 0.05). As the frequency of nocturnal LH secretory bursts increased, the frequency of testosterone bursts decreased (r = -0.746, P = 0.034). We conclude that healthy aging is associated with diminished nocturnal testosterone production attributable to 1) a decrement in the mass of testosterone released per burst and 2) an inadequate response by the pituitary gland to enhance testosterone production via an accelerated LH secretory pulse frequency. We speculate that this may reflect a partial desensitization of Leydig cells to LH.

Adult↗
PubMedDate not supplied

Three-dimensional chromatin distribution in neuroblastoma nuclei shown by confocal scanning laser microscopy.

The relationship between cell shape and function has long been of interest. However, although the behaviour of the cytoskeleton during the cell cycle has been studied extensively variations in the shape and three-dimensional substructure of the nucleus are less well documented. The spatial distribution of chromatin has previously been studied by a mathematical analysis of the optical densities of stained nuclei, allowing an indirect derivation of the three-dimensional distribution of chromatin. More direct information on chromatin organization can be obtained from electron-microscopic serial sections, although this is very laborious. Using an iterative deconvolution algorithm, Agard and Sedat achieved a degree of optical sectioning in conventional fluorescence microscopy and reconstructed the three-dimensional arrangement of polytene chromosomes. We report here on the three-dimensional structure of cultured mammalian cells as visualized by confocal scanning laser microscopy (CSLM). The exceptionally short depth of field of this imaging technique provides direct optical sectioning which, together with its higher resolution, makes CSLM extremely useful for studying the three-dimensional morphology of biological structures.

Anaphase↗
PubMed2004

Cluster computing for digital microscopy.

Microscopy is becoming increasingly digital and dependent on computation. Some of the computational tasks in microscopy are computationally intense, such as image restoration (deconvolution), some optical calculations, image segmentation, and image analysis. Several modern microscope technologies enable the acquisition of very large data sets. 3D imaging of live cells over time, multispectral imaging, very large tiled 3D images of thick samples, or images from high throughput biology all can produce extremely large images. These large data sets place a very large burden on laboratory computer resources. This combination of computationally intensive tasks and larger data sizes can easily exceed the capability of single personal computers. The large multiprocessor computers that are the traditional technology for larger tasks are too expensive for most laboratories. An alternative approach is to use a number of inexpensive personal computers as a cluster; that is, use multiple networked computers programmed to run the problem in parallel on all the computers in the cluster. By the use of relatively inexpensive over-the-counter hardware and open source software, this approach can be much more cost effective for many tasks. We discuss the different computer architectures available, and their advantages and disadvantages.

Cells↗
PubMed2004

Ligand-modified vesicular stomatitis virus glycoprotein displays a temperature-sensitive intracellular trafficking and virus assembly phenotype.

The production of potentially targetable VSV-G-pseudotyped retrovirus vectors has been hampered by inadequate understanding of the structure-function relationships of the VSV-G protein. In these studies we demonstrate assembly and production of MLV-based and HIV-1-based vector particles using VSV-G proteins modified by the insertion of a peptide ligand into a site corresponding to amino acid position 24 of the native VSV-G molecule. The inserted ligand represents the decapeptide encoding the collagen-binding domain of von Willebrand factor. We have used deconvolution microscopy to demonstrate that the modified VSV-G molecules sequester in perinuclear structures and are unavailable for assembly of infectious virus particles at the cell surface under standard tissue culture conditions at 37 degrees C. In contrast, at a lower permissive temperature of 30 degrees C, the modified VSV-G protein traffics appropriately to the cell surface and participates in useful titers. Furthermore, VSV-G-pseudotyped MLV-based and HIV-1-based vectors displaying the collagen-binding domain demonstrate a statistically significant increased attachment to a collagen matrix as indicated by an ELISA-like cell binding assay and by a focus transduction assay.

Amino Acid Sequence↗
PubMed2002

In vivo release of bupivacaine from subcutaneously administered oily solution. Comparison with in vitro release.

A non-randomized cross-over study was performed with bupivacaine HCl (5 mg x ml(-1)) aqueous solution and bupivacaine free base (4.44 mg x ml(-1)) in Viscoleo/castor oil 2:1 (v/v) administered s.c. to male Wistar rats. Plasma levels were analyzed by LC-MS. Plasma profiles obtained after administration of oily solution showed a prolonged bupivacaine release with lower peak plasma levels as compared to administration of an aqueous formulation applied in the same compartment. t(1/2), t(max), C(max) and AUC(0-infinity) for the aqueous solution were 63+/-8 min, 19+/-16 min, 194+/-46 ng x ml(-1) and 25,000+/-3000 ng min x ml(-1), respectively, while the corresponding data for the oil solution were 368+/-89 min, 334+/-186 min, 36+/-25 ng x ml(-1) and 25,000+/-6000 ng x min x ml(-1). The present data indicate the potential of designing an oil formulation of bupivacaine with a prolonged local analgetic effect exhibiting a minimum of systemic toxicity. In vivo release of bupivacaine from the oil solution was evaluated by a numerical deconvolution method. In vivo release kinetics was found to be first-order and corresponded well with in vitro release kinetics found using a rotating dialysis cell. This led to establishment of an in vitro/in vivo correlation for this particular formulation.

Anesthetics, Local↗
PubMed2004

Alterations in mitochondrial membrane potential during preimplantation stages of mouse and human embryo development.

Mitochondria are cellular organelles regulating metabolism and cell death pathways. This study examined changes in mitochondrial membrane potential (deltapsim) throughout the stages of preimplantation development in mouse embryos conceived either in vivo or in vitro and human embryos donated to research from IVF. Embryos stained with the deltapsim-sensitive dye (JC-1) were quantified for the ratio of high- to low-polarized mitochondria using a deconvolution microscope. Overall, mouse zygotes and early embryos contain a subset of high-polarized mitochondria with a progressive increase in the ratio of deltapsim observed with increasing cleavage. A transient increase in the ratio of high to low deltapsim was observed in in vivo fertilized 2-cell stage embryos, coincident with embryonic genome activation in the mouse, but not in 2-cell embryos obtained through IVF. We further observed that arrested mouse 2-cell embryos possessed an increased ratio of deltapsim compared with non-arrested embryos. In human 8-cell embryos we observed an increased ratio of high- to low-polarized mitochondria with increasing degrees of embryo fragmentation. We concluded that the pattern of mitochondrial membrane potential progressively changes throughout preimplantation development, and that an aberrant shift in deltapsim could contribute to, or is associated with, decreased developmental potential.

Animals↗
PubMed1996

Contrasts in the gonadotropin-releasing hormone dose-response relationships for luteinizing hormone, follicle-stimulating hormone and alpha-subunit release in young versus older men: appraisal with high-specificity immunoradiometric assay and deconvolution analysis.

The secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) is regulated by gonadotropin-releasing hormone (GnRH). As men age, mean serum concentrations of immunoreactive gonadotropic hormones tend to increase, while serum testosterone concentrations tend to decline. To evaluate age-related changes in gonadotroph cell function, we have assessed the dose-dependent secretory responses of immunoreactive LH, FSH and alpha-subunit to saline versus five doses of GnRH in older and young men. Ten older men, mean age 66 years (range 61-78), and nine young men, mean age 26 years (range 22-30), received iv bolus injections of GnRH (range 10-100 micrograms) in randomized order every 2 h, except that the 100-microgram dose was always given last. Blood samples for immunoradiometric assays of serum LH, FSH and alpha-subunit concentrations were obtained every 10 min for a total of 12 h, which included a 2-h preinjection baseline. Deconvolution analysis was performed to estimate gonadotropin and alpha-subunit secretory burst mass, amplitude and duration, as well as endogenous LH, FSH and alpha-subunit half-lives. The mean (+/- SEM) baseline 2-h serum FSH (IU/I) concentration was higher in older than younger men (5.9 +/- 0.8 vs 3.8 +/- 0.5, p < 0.05). The mean 2-h serum LH concentrations after GnRH were significantly higher than corresponding values in young men at GnRH doses of 25, 50 and 75 micrograms, and in the case of FSH at GnRH doses of 10 and 25 micrograms. Non-linear curve-fitting of these dose-response relationships revealed that the calculated maximal mean 2-h serum LH concentration response (IU/l) was higher in older than young men following GnRH stimulation: 15.4 (13.5-16.2) vs 10.8 (8.7-12.1) (95% confidence interval). The maximal mean 2-h serum FSH concentration response (IU/l) was also significantly higher in older men: 11.9 (10.2-13.1) versus 8.6 (7.2-9.6). Maximal alpha-subunit responses (microgram/l) were similarly increased in the older cohort: 1.16 (0.99-1.25) vs 0.83 (0.71-0.91). The incremental LH (p < 0.05) and FSH (p < 0.01) secretory burst mass from 10 to 25 micrograms GnRH was significantly greater in older than younger men. The LH and FSH half-lives and second component alpha-subunit half-lives were similar in older and young men. In addition, secretory burst durations were invariant of age. In contrast, by non-linear curve-fitting, the calculated mass of LH secreted was higher in older men at 13.5 (11.8-15) vs 10.6 (9.2-11.7) IU/l of distribution volume (p < 0.05) for the maximal absolute mass and 11.3 (9.5-12.7) vs 7.4 (6.0-8.4) IU/l (p < 0.05) for the maximal incremental mass of LH secreted after GnRH. The estimated maximal mass of FSH secreted after GnRH also was higher in older men: 4.6 (3.4-5.5) vs 3.2 (2.9-3.4) IU/l (p < 0.01). Finally, calculated maximal GnRH-stimulated alpha-subunit secretory burst mass was statistically greater in older individuals: 2.3 (1.8-2.5) vs 1.6 (1.4-1.8) micrograms/l. In contrast, half-maximally effective GnRH doses were not different in the two age groups. We conclude that older men show significantly increased maximal and incremental gonadotropin release due to amplified secretory burst mass in response to escalating doses of GnRH with no evident differences in LH, FSH, or alpha-subunit half-lives or secretory burst durations. Increased gonadotroph responsiveness may be due to diminished gonadal hormone negative feedback or primary alterations in the hypothalamo-pituitary unit with aging.

Adult↗
PubMed1984

A video image correlation technique for the measurement of electrophoretic mobility.

The electrophoretic movement of blood cells has been examined with a video image correlator using the signal from a 625 line monochrome television camera attached to the optical arrangement. The diffraction pattern of a cell produces a characteristic signal which can be detected and registered in any television scan line to form the data of the first image frame. Under the influence of an electric field, cells moved across the television field of view and after a short interval appeared on different scan lines. The detection and registering process was repeated, and a cross-correlation function between the first and second image frame was computed. This function was stored in an output buffer which was continuously updated as fresh functions were integrated with the existing contents. The mean electrophoretic mobility of a suspension of particles can be calculated from the peak position of the integrated correlation function. Using a triangle deconvolution procedure, the relative contributions of subpopulations of lymphocytes prepared from peripheral blood samples were examined.

Blood Cells↗
PubMed1996

Suprachiasmatic nucleus of the human brain: an immunocytochemical and morphometric analysis.

BACKGROUND: The present paper describes the immunocytochemical and morphometric characteristics of two major cell groups of the suprachiasmatic nucleus (SCN) in the human hypothalamus: the vasopressin (VP) and vasoactive intestinal polypeptide (VIP) neuronal subdivisions. The dimensions (volume and length) and the number of neurons expressing each peptide in the two subdivisions were obtained, as well as the mean diameter of the cell nuclei. All morphometric parameters were studied in relation to sex and age. METHODS: Brains of 42 human subjects (22 males and 20 females) ranging in age from 10 to 92 years were obtained at autopsy. The hypothalamic area containing the SCN was dissected from each brain, dehydrated, and embedded in paraffin. Serial sections of 6 microns were cut in a coronal plane and stained with thionin for general orientation. To determine the architectonic boundaries of the VP- and VIP-expressing cell populations every 25th section was immunocytochemically stained by means of antibodies against arginine VP or VIP using the peroxidase-antiperoxidase method. The VP- and VIP-expressing cell numbers in the SCN of each subject were estimated by unilaterally counting the number of nuclear profiles with the aid of a Zeiss microscope under x 500 magnification, using a deconvolution procedure and a correction for section thickness. RESULTS: The main portion of the VP positive neurons is located in the dorsomedial part of the SCN and is rostrocaudally longer in females than in males (1.76 +/- 0.12 mm and 1.40 +/- 0.10 mm, respectively). The volume of the VP subdivision is 0.244 +/- 0.017 mm3 and contains 6,890 +/- 520 VP-immunoreactive neurons, with a mean density of about 29,000 neurons/mm3. No significant sexual dimorphism or age-related alterations in the population of VP neurons is found. The VIP positive neurons are mainly located in the ventral and central part of the SCN and extend rostrocaudally in a similar way in females and males (1.07 +/- 0.08 mm and 1.02 +/- 0.11 mm, respectively). The volume of the VIP subdivision is 0.034 +/- 0.004 mm3 and contains 1,700 +/- 140 VIP-immunoreactive neurons, with a mean density of about 63,000 neurons/mm3. An age-dependent sexual dimorphism is observed in the number of VIP-expressing neurons in the SCN: young males have about twice as many VIP neurons as females of the same age, whereas in middle-aged subjects this sexual difference is reversed, and less robust, with females now having about 1.7 times as many VIP neurons as males. In old subjects the difference in VIP cell number between men and women disappears. CONCLUSIONS: The present study clearly shows that the population of VP neurons in the human SCN is considerably larger than the population of VIP neurons. Furthermore, the age-related sexual differences in the VIP cell number reinforces the idea that the SCN is not only involved in the timing of circadian rhythms but also in the temporal organization of reproductive functions.

Adolescent↗
PubMed2003

A method for dynamic spectrophotometric measurements in vivo using principal component analysis-based spectral deconvolution.

A method was developed for dynamic spectrophotometric measurements in vivo in the presence of non-specific spectral changes due to external disturbances. This method was used to measure changes in mitochondrial respiratory pigment redox states in photoreceptor cells of live, white-eyed mutants of the blowfly Calliphora vicina. The changes were brought about by exchanging the atmosphere around an immobilised animal from air to N2 and back again by a rapid gas exchange system. During an experiment reflectance spectra were measured by a linear CCD array spectrophotometer. This method involves the pre-processing steps of difference spectra calculation and digital filtering in one and two dimensions. These were followed by time-domain principal component analysis (PCA). PCA yielded seven significant time domain principal component vectors and seven corresponding spectral score vectors. In addition, through PCA we also obtained a time course of changes common to all wavelengths-the residual vector, corresponding to non-specific spectral changes due to preparation movement or mitochondrial swelling. In the final step the redox state time courses were obtained by fitting linear combinations of respiratory pigment difference spectra to each of the seven score vectors. The resulting matrix of factors was then multiplied by the matrix of seven principal component vectors to yield the time courses of respiratory pigment redox states. The method can be used, with minor modifications, in many cases of time-resolved optical measurements of multiple overlapping spectral components, especially in situations where non-specific external influences cannot be disregarded.

Animals↗
PubMed2004

In vivo analysis of synaptonemal complex formation during yeast meiosis.

During meiotic prophase a synaptonemal complex (SC) forms between each pair of homologous chromosomes and is believed to be involved in regulating recombination. Studies on SCs usually destroy nuclear architecture, making it impossible to examine the relationship of these structures to the rest of the nucleus. In Saccharomyces cerevisiae the meiosis-specific Zip1 protein is found throughout the entire length of each SC. To analyze the formation and structure of SCs in living cells, a functional ZIP1::GFP fusion was constructed and introduced into yeast. The ZIP1::GFP fusion produced fluorescent SCs and rescued the spore lethality phenotype of zip1 mutants. Optical sectioning and fluorescence deconvolution light microscopy revealed that, at zygotene, SC assembly was initiated at foci that appeared uniformly distributed throughout the nuclear volume. At early pachytene, the full-length SCs were more likely to be localized to the nuclear periphery while at later stages the SCs appeared to redistribute throughout the nuclear volume. These results suggest that SCs undergo dramatic rearrangements during meiotic prophase and that pachytene can be divided into two morphologically distinct substages: pachytene A, when SCs are perinuclear, and pachytene B, when SCs are uniformly distributed throughout the nucleus. ZIP1::GFP also facilitated the enrichment of fluorescent SC and the identification of meiosis-specific proteins by MALDI-TOF mass spectroscopy.

Blotting, Western↗